Original Article
Suppression of transient receptor potential melastatin 4 expression promotes conversion of endothelial cells into fibroblasts via transforming growth factor/activin receptor-like kinase 5 pathway Cesar Echeverrı´a a,b,c, Ignacio Montorfano a,c, Claudio Cabello-Verrugio d, Ricardo Armise´n b,e, Diego Varela b,f, and Felipe Simon a,g
Objective: To study whether transient receptor potential melastatin 4 (TRPM4) participates in endothelial fibrosis and to investigate the underlying mechanism. Methods: Primary human endothelial cells were used and pharmacological and short interfering RNA-based approaches were used to test the transforming growth factor beta (TGF-b)/activin receptor-like kinase 5 (ALK5) pathway participation and contribution of TRPM7 ion channel. Results: Suppression of TRPM4 expression leads to decreased endothelial protein expression and increased expression of fibrotic and extracellular matrix markers. Furthermore, TRPM4 downregulation increases intracellular Ca2þ levels as a potential condition for fibrosis. The underlying mechanism of endothelial fibrosis shows that inhibition of TRPM4 expression induces TGF-b1 and TGF-b2 expression, which act through their receptor, ALK5, and the nuclear translocation of the profibrotic transcription factor smad4. Conclusion: TRPM4 acts to maintain endothelial features and its loss promotes fibrotic conversion via TGF-b production. The regulation of TRPM4 levels could be a target for preserving endothelial function during inflammatory diseases.
INTRODUCTION
A
cute and chronic systemic inflammation is a complex pathological condition that is associated with a high morbidity and mortality rate. Several diseases are associated with acute and chronic systemic inflammation, including hypertension, atherosclerosis, obesity, pulmonary disease, neurodegeneration, diabetes, and sepsis [1–5]. This condition is characterized by an overactivation of the immune system, including immune cell activation and the oversecretion of proinflammatory cytokines. To date, substantial basic and clinical efforts have been directed toward improving the current therapies against these diseases. However, despite the advances made in the last two decades, numerous improvement remains to be achieved. During systemic inflammation, cytokines such as tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), IL-1b, and transforming growth factor beta (TGF-b) circulate through the bloodstream. Thus, these cytokines inevitably interact with endothelial cells on the inner walls of blood vessels. Given that endothelial cells express cytokine receptors, these molecules trigger intracellular signaling pathways that maintain and enhance the inflammatory response [6,7]. It has been demonstrated that the pro-inflammatory cytokine TGF-b, including its isoforms TGF-b1 and
Keywords: endothelial dysfunction, fibrosis, inflammation, TGF-b, TRPM4 Abbreviations: a-SMA, a-smooth muscle actin; ALK5, activin receptor-like kinase 5; Col, collagen; ECM, extracellular matrix; EndMT, endothelial-to-mesenchymal transition; FSP-1, fibroblast-specific protein; HUVECs, human umbilical vein endothelial cells; IL, interleukin; siRNA, short interfering RNA; TbR, TGF-b receptor; TGF-b, transforming growth factor-beta; TNF-a, tumor necrosis factor-alpha; TRPM4, transient receptor potential melastatin 4
Journal of Hypertension 2015, 33:981–992 a Laboratorio de Fisiopatologı´a Integrativa, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Facultad de Medicina, Universidad Andres Bello, b Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, c Laboratorio de Bionanotecnologia, Universidad Bernardo O’Higgins, dLaboratorio de Biologı´a y Fisiopatologı´a Molecular, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Facultad de Medicina, Universidad Andres Bello, eCentro de Investigacion y Tratamiento del Cancer, fCentro de Estudios Moleculares de la Celula and gMillennium Institute on Immunology and Immunotherapy, Santiago, Republic of Chile
Correspondence to Felipe Simon, Departamento de Ciencias, Biologicas, Facultad de Ciencias Biologicas, Facultad de Medicina, Universidad Andres Bello, Av. Republica 252, 8370134, Santiago, Republic of Chile. Tel: +562 2661 5653; fax: +562 2698 0414; e-mail:
[email protected] Received 20 July 2014 Revised 17 November 2014 Accepted 17 November 2014 J Hypertens 33:981–992 ß 2015 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0000000000000496
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TGF-b2, induces the conversion of endothelial cells into activated fibroblasts, also known as myofibroblasts, through a process known as the endothelial-to-mesenchymal transition (EndMT) [8,9]. TGF-b isoforms act by binding to the TGF-b receptor type II (TbRII), which recruits TbRI, or activin receptor-like kinase 5 (ALK5). Subsequently, ALK5 phosphorylates smad2/3, which bind smad4 to regulate target gene transcription and promote fibrosis [8,9]. The cytokines TNF-a, IL-6, and IL-1b also induce EndMT [10–12]. Interestingly, IL-1b enhances both TGF-b2induced and TNF-a-induced EndMT [11–13]. In line with these findings, we recently showed that the endotoxin lipopolysaccharide is able to promote endothelial fibrosis [14]. EndMT is characterized by the switch from an endothelial protein expression profile to a fibrotic-like pattern. During EndMT, the endothelial markers CD31 and VE-cadherin were downregulated in endothelial cells, whereas the fibroblast-specific gene a-smooth muscle actin (a-SMA) and the fibroblast-specific protein 1 (FSP-1) were upregulated. In addition, the expression and secretion of extracellular matrix (ECM) proteins such as fibronectin and collagen (Col) are highly increased [8–14]. Thus, proinflammatory cytokines and other inflammatory mediators induce endothelial fibrosis, along with the resulting endothelial impairment. Changes in intracellular calcium concentrations are essential for the fibrotic program. Extracellular Ca2þ chelation was sufficient to inhibit the proliferation of myofibroblasts from cultured rat cardiac fibroblasts [15,16]. Furthermore, the inhibition if Ca2þ influx attenuates the main attributes of fibrosis, such as increased intracellular oxidative stress and proinflammatory cytokines synthesis and secretion [15–17]. In line with this, increased cell migration, a major feature of myofibroblast, is also dependent on intracellular Ca2þ changes [18,19]. Correspondingly, changes in intracellular Ca2þ concentration modulate liver fibrosis [20]. Hence, intracellular Ca2þ regulation is crucial for fibrosis to develop. As a result of its therapeutic implications, the identification of the molecular entity that controls intracellular Ca2þ levels during fibrogenesis is a question of great significance. Several types of calcium channels are known to be involved in generating fibrosis. L-type calcium channels modulate renal perivascular fibrosis [21]. Similarly, it has been shown that the inhibition of T-type and L-type calcium channels decreases tubulointerstitial fibrosis [22]. Recently, we reported that the transient receptor potential melastatin 7 calcium channel is crucially involved in endotoxin-induced endothelial fibrosis [23]. In congruence, calcium channel blockers decreased cardiac fibrosis in addition to complementary treatments and attenuated liver fibrogenesis [24–26]. Thus, these data indicate that calcium channels are involved in controlling intracellular Ca2þ levels to promote fibrosis. The transient receptor potential melastatin 4 (TRPM4) channel is a Ca2þ-activated nonselective cation channel that is permeable to monovalent cations such as Naþ and Kþ but is virtually impermeable to divalent ions such as Ca2þ and Mg2þ [27,28]. TRPM4 is also modulated by ROS and it is expressed in several cell types, including arterial and 982
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vein-derived endothelial cells [29–31]. TRPM4 performs a number of cell functions, including the regulation of intracellular Ca2þ overloading, the preservation of plasma membrane potential, and the control of intracellular Ca2þ oscillations [27,28,32]. TRPM4 is involved in the immune response during inflammation. TRPM4 plays differential functions in Th1 and Th2 cells through regulating Ca2þ signaling and the nuclear localization of NFATc1. Additionally, TRPM4 controls the migration of bone-marrowderived mast cells (BMMCs) and dendritic cells by regulating Ca2þ-dependent cytoskeleton rearrangement [33–35]. In a sepsis transient receptor potential melastatin-4-knockout (TRPM4-KO) mice model has been reported that the mortality is increased, suggesting the crucial role for TRPM4-dependent Ca2þ regulation on immune cell activation [36]. On the other hand, TRPM4-KO mice showed a decreased fragmentation of capillaries and small vessels usually observed when a spinal cord injury is produced [37], suggesting a protective role for TRPM4 suppression on inflammatory conditions. Taken together, the TRPM4 ion channel appears to be a potential regulator of intracellular Ca2þ concentrations during inflammatory conditions. However, the role of TRPM4 in endothelial fibrosis is not known. Therefore, the aim of this article was to investigate whether TRPM4 expression participates in the progression of endothelial fibrosis. We demonstrate that the inhibition of TRPM4 expression increases the intracellular Ca2þ levels. Suppressing TRPM4 expression induces an increase in fibrotic markers and a decrease in endothelial-specific proteins and also induces increases in ECM proteins, thereby promoting the conversion of endothelial cells into myofibroblasts. Further elucidation of the underlying mechanisms revealed that the inhibition of TRPM4 expression promotes the expression and secretion of the endothelial fibrosis inducers TGF-b1 and TGF-b2. In line with this finding, the conversion of endothelial cells into activated fibroblasts induced by TRPM4 downregulation was dependent on ALK5 activity. Furthermore, the increased intracellular Ca2þ levels observed upon the suppression of TRPM4 expression were independent of ALK5 activation. Interestingly, TRPM4 expression inhibition induced the nuclear translocation of the profibrotic transcription factor smad4. Taken together, these results show that TRPM4 ion channel is a key regulator of the endothelial phenotype, wherein the downregulation of TRPM expression promotes conditions that are suitable for the establishment of fibrosis. Thus, maintaining TRPM4 expression at appropriate levels would be beneficial for the control of endothelial dysfunction during systemic inflammation and other inflammatory diseases.
MATERIALS AND METHODS Details of all procedures are provided in the supplementary digital content, http://links.lww.com/HJH/A458.
Primary cell culture Human umbilical vein endothelial cells (HUVECs) were isolated from freshly obtained umbilical cord veins from Volume 33 Number 5 May 2015
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TRPM4 suppression-induced endothelial fibrosis
normal pregnancies, after patient’s informed consent. The investigation conforms with the principles outlined in the Declaration of Helsinki. The Commission of Bioethics and Biosafety of Universidad Andres Bello approved all experimental protocols. Cells were grown in gelatin-coated dishes at 378C in a 5%:95% CO2:air atmosphere in medium 199 (Sigma, St Louis, MO, USA St Louis, Missouri, USA), containing 100 mg/ml endothelial cell growth supplement (ECGS; (Sigma), 100 mg/ml heparin, 5 mmol/l D-glucose, 3.2 mmol/l L-glutamine, 10% fetal bovine serum (FBS; GIBCO, NY), and 50 U/ml penicillin-streptomycin (Sigma-Aldrich, St Louis, Missouri, USA).
Small interfering RNA and transfection SiGENOME SMART pool short interfering RNA (siRNA; four separated siRNAs per human TRPM4 transcript) were purchased from Dharmacon (Dharmacon, Lafayette, CO, USA). The following siRNA were used: human TRPM4 (siRNA-TRPM4) and nontargeting siRNA (siRNA-control (CTRL) knock-out) used as a control. In brief, HUVECs were transfected with 5 nM siRNA using DharmaFECT 4 transfection reagent (Dharmacon) used according to the manufacturer’s protocols in serum-free medium.
Measurement of intracellular calcium levels Primary endothelial cells were nontransfected (wild-type) or transfected with siRNA-CTRL or siRNA-TRPM4 for 72 h. Experiments were performed as previously described [23,30,38]. Briefly, endothelial cells were harvested, washed twice, resuspended, and loaded with either 5 mmol/l Fluo-4 or 15 mmol/l Fura-Red calcium-sensitive cell permeant dyes (all from Invitrogen). The labeled cells were then analyzed immediately by flow cytometry.
Quantitative real-time PCR and western blot procedures Equal amounts of RNA were used as templates in each reaction. QPCR was performed using the SYBR Green PCR Master Mix (AB Applied Biosystems, Foster City, California, USA). Assays were run using an Eco Real-Time PCR System (Illumina, San Diego, California, USA). Data are presented as relative mRNA levels of the gene of interest normalized to relative levels of 28s mRNA. Endothelial cells transfected with siRNA-CTRL or siRNA-TRPM4 were lysed in cold lysis buffer, and then proteins were extracted. Supernatants were collected and stored in the same lysis buffer. Protein extract and supernatant were subjected to SDS-PAGE and resolved proteins were transferred to a nitrocellulose or poly vinylidene fluoride (PVDF) membrane. The blocked membrane was incubated with the appropriate primary antibody, washed twice, and incubated with a secondary antibody. Bands were revealed using a peroxidase-conjugated IgG antibody. Tubulin was used as a loading control. For a detailed list of antibodies used, see Table S1, http:// links.lww.com/HJH/A458.
Fluorescent immunocytochemistry Endothelial cells transfected with siRNA-CTRL or siRNATRPM4 were washed twice with PBS and fixed. The cells were subsequently washed again and incubated with the primary antibodies. Then, cells were washed twice and Journal of Hypertension
incubated with the secondary antibodies. Samples were mounted with ProLong Gold antifade mounting medium with 4’-6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, California, USA). For a detailed list of antibodies used, see Table S2, http://links.lww.com/HJH/A458.
Reagents Fluo-4 and Fura-Red were purchased from Invitrogen. SB431542 were purchased from Tocris (Ellisville, Missouri, USA). Human TGF-b1 and TGF-b2 were purchased from R&D Systems (Minneapolis, Minnesota, USA). Buffers and salts were purchased from Merck Biosystems (Darmstadt, Germany).
Data analysis All results are presented as means SD. Statistical differences were assessed by Student’s t-test (Mann–Whitney) or one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test and considered significant at P less than 0.05.
RESULTS Intracellular Ca2þ levels increase upon the suppression of transient receptor potential melastatin 4 expression in endothelial cells The initiation and development of fibrotic processes are characterized by increases in the intracellular Ca2þ concentration ([Ca2þ]i) [15,16,20]. Therefore, we studied whether the downregulation of TRPM4 modified the basal levels of [Ca2þ]i in order to establish conditions suitable for generating fibrosis. To that end, we monitored changes in the intracellular calcium level by means of two different Ca2þ-sensitive fluorescent dyes: Fluo-4 and Fura-Red. We used molecular biology approach to downregulate TRPM4 expression. A specific siRNA against the human isoform of TRPM4 (siRNA-TRPM4) was used. TRPM4 channel expression was downregulated with more than 95% efficiency using this siRNA, compared to endothelial cells transfected with a nontargeting sequence siRNA used as a control (siRNA-CTRL; Fig. 1a and b). Endothelial cells transfected with siRNA-CTRL showed similar Fluo-4 and Fura-Red fluorescence levels as those observed in nontransfected cells (Fig. 1c and d), reflecting similar calcium levels. However, endothelial cells transfected with siRNA-TRPM4 showed an increased Fluo-4 fluorescence signal, indicating increased [Ca2þ]i (Fig. 1c). Similarly, siRNA-TRPM4-transfected endothelial cells showed decreased fluorescence from Fura-Red, which decreases its fluorescence upon binding calcium, also indicating that intracellular calcium was increased (Fig. 1d). These results suggest that the suppressing TRPM4 expression increases the [Ca2þ]I, generating an appropriate scenario for fibrosis development.
Inhibiting transient receptor potential melastatin 4 expression in endothelial cells induces a fibroblast-like phenotype Endothelial cells cultured in the presence of vehicle showed a round, short-spindle morphology with a cobblestone appearance (Fig. 1e). In contrast, endothelial cells www.jhypertension.com
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FIGURE 1 Changes in the intracellular calcium and the fibrotic-like endothelial morphology induced by the inhibition of TRPM4 expression in endothelial cells. (a and b) TRPM4 expression downregulation by siRNA. ECs were transfected with a specific siRNA against the human TRPM4 isoform (siRNA-TRPM4) or a nontargeting siRNA [siRNA-control (CTRL)]. (a) Representative images from western blot experiments performed to detect TRPM4 in cells transfected with siRNA-CTRL or siRNA-TRPM4. (b) Densitometric analyses of several experiments, as shown in (a). Protein levels were normalized against tubulin, and the data are expressed relative to cells transfected with siRNA-CTRL (n ¼ 5). Statistical significance was assessed using Student’s t-test (Mann–Whitney). P < 0.001. Graph bars show the mean SD. (c and d) Calcium overloading was evaluated using the Ca2þ-sensitive fluorescent dyes Fluo-4 (c) and Fura-Red (d) in nontransfected ECs [wild type (WT)] and in ECs transfected with siRNACTRL or siRNA-TRPM4 (n ¼ 5). Statistical significance was assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis) followed by Dunn’s post hoc test. P < 0.05; P < 0.01. Graph bars show the mean SD. (e–l) Morphological changes resembling endothelial fibrosis in human ECs. Figures show representative phase-contrast images from at least three separates experiments with nontransfected ECs exposed to vehicle (e), 10 ng/ml TGF-b1 (f), 2.5 ng/ml TGF-b2 (g), ECs transfected with siRNA-CTRL (h) for 72 h, and ECs transfected with siRNA-TRPM4 for 48 (i) or 72 h (j). Scale bar represents 50 mm, n ¼ 3–4. (k) Normalized endothelial cell length distribution of nontransfected ECs exposed to vehicle (filled bars), 10 ng/ml TGF-b1 (dashed bars), and 2.5 ng/ml TGF-b2, (dashed bars), as well as ECs transfected with siRNA-CTRL (grey bars), and ECs transfected with siRNA-TRPM4 (empty bars), n ¼ 4–5. (l) Normalized endothelial cell length in which length/width greater than 2 for nontransfected ECs exposed to vehicle, 10 ng/ml TGF-b1, and 2.5 ng/ml TGF-b2, ECs transfected with siRNA-CTRL and ECs transfected with siRNA-TRPM4 (n ¼ 4–5). Statistical significance was assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis) followed by Dunn’s post hoc test. P < 0.01. Graph bars show the mean SD. ECs, endothelial cells; siRNA, short interfering RNA; TRPM4, transient receptor potential melastatin 4.
exposed to TGF-b1, the most studied EndMT inducer, showed a spindle-shaped phenotype typical of fibroblasts (Fig. 1f), indicating the occurrence of an EndMT process. 984
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TGF-b2 similarly induced the conversion into a fibroblast morphology (Fig. 1g). Endothelial cells transfected with siRNA-CTRL exhibited a similar morphology as that Volume 33 Number 5 May 2015
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FIGURE 2 Suppression of TRPM4 expression induces changes in endothelial, fibrotic, and ECM proteins. ECs were transfected with a specific siRNA against the human TRPM4 isoform (siRNA-TRPM4) or a nontargeting siRNA [siRNA-control (CTRL)], and protein expression was analyzed. (a, c, e, g, i, and k) Representative images from western blot experiments performed to detect CD31 (a), VE-cadherin (VE-Cad) (c), a-SMA (e), FSP-1 (g), FN (i), and Col III (k). (b, d, f, h, j, and l) Densitometric analyses of the experiments shown in (a, c, e, g, i, and k,
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observed in nontransfected endothelial cells (Fig. 1h), whereas endothelial cells transfected with siRNA-TRPM4 underwent morphological changes similar to those observed in the presence of TGF-b1 or TGF-b2, suggesting that TRPM4 downregulation could be triggering a similar fibroblast conversion process (Fig. 1i and j). To study these phenotypic changes in detail, we measured the length of cells in each condition, showing a differential distribution in cell length. The mean cell lengths of endothelial cells exposed to TGF-b1 and TGF-b2 were approximately three-fold higher than that observed in vehicle-treated cells. Similarly, endothelial cells transfected with siRNA-TRPM4 exhibited an analogous (3-fold) increase in the cell length distribution. As we expected, siRNA-CTRL-transfected cells were similar to vehicle-treated cells (Fig. 1k). Next, we counted cells whose length was at least twice their width (length/width >2). The results showed that the lengths of endothelial cells exposed to TGF-b1 and TGF-b2, as well as endothelial cells transfected with siRNA-TRPM4, were approximately 10-fold higher than siRNA-CTRL-transfected cells or vehicle-treated cells (WT; (Fig. 1l). The enrichment of our endothelial cultures was tested by performing a detailed examination with VE-cadherin used as a specific endothelial marker. We found that over 99% of cells in our endothelial cell cultures were positive for VE-cadherin, demonstrating that our primary endothelial cell cultures were highly enriched in endothelial cells, without potential contamination from fibroblasts or mesenchymal-like cells (Supplemental Figure S1, http://links.lww.com/HJH/A458).
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Endothelial cells transfected with siRNA-TRPM4 showed a decrease in the expression of endothelial markers compared with siRNA-CTRL-transfected endothelial cells. siRNA-TRPM4-transfected endothelial cells showed a decrease in the expression of the endothelial proteins CD31 (Fig. 2a and b) and VE-cadherin (Fig. 2c and d). Furthermore, endothelial cells transfected with siRNATRPM4 showed an increase in the expression of fibroticspecific genes compared with cells transfected with siRNACTRL. The expression of the fibrotic markers a-SMA (Fig. 2e and f) and FSP-1 (Fig. 2g and h) was significantly increased in cells transfected with siRNA-TRPM4. Similar results were observed using the selective TRPM4 blocker 9-phenantrol (Supplemental Figure S2, http://links.lww.com/HJH/A458) [39]. As ECM protein accumulation is a decisive step in the progression of fibrogenesis, we were prompted to measure the fibronectin and collagen protein levels in the supernatants of endothelial cell cultures. Endothelial cells respectively). Protein levels were normalized against tubulin, and data are expressed relative to siRNA-CTRL-transfected cells (n ¼ 4–6). Statistical significance was assessed using Student’s t-test (Mann–Whitney). P < 0.01. Graph bars show the mean SD. ECs, endothelial cells; FM, fibronectin; siRNA, short interfering RNA; SMA, smooth muscle actin; TRPM4, transient receptor potential melastatin 4.
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Next, we studied the changes induced by TRPM4 downregulation in the cellular localization and distribution of endothelial and fibrotic proteins using immunocytochemistry experiments. As expected, endothelial cells transfected with siRNA-CTRL showed typical CD31 labeling, localized predominantly to the plasma membrane, whereas a-SMA was weakly expressed (Fig. 3a and b). Additionally, VEcadherin labeling was detected at the plasma membrane, whereas FSP-1 expression was almost undetectable (Fig. 3c and d). In contrast, endothelial cells transfected with
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TGF-b1 and TGF-b2 expression and secretion are stimulated by transient receptor potential melastatin 4 downregulation in endothelial cells As mentioned previously, TGF-b1 and TGF-b2 has been well studied as EndMT inducers [8,9]. In line with these siRNA-CRTL (i)
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Suppressing transient receptor potential melastatin 4 expression induces the subcellular redistribution of endothelial, fibrotic and extracellular matrix proteins in endothelial cells
siRNA-TRPM4 displayed increased a-SMA labeling in fibrotic-like stress fibers and decreased CD31 expression (Fig. 3e and f). Furthermore, FSP-1 labeling was greatly increased in the intracellular compartment, whereas VE-cadherin was virtually absent (Fig. 3g and h). To investigate the effect produced by TRPM4 downregulation on the cellular localization and distribution of ECM proteins, we carried out immunocytochemistry experiments. Endothelial cells transfected with siRNA-CTRL showed a typical CD31 (Fig. 3i and j) and VE-cadherin (Fig. 3k and l) labeling, which was restricted to the plasma membrane, whereas fibronectin (Fig. 3i–l) was expressed at low levels. In contrast, endothelial cells transfected with siRNA-TRPM4 showed increased fibronectin labeling (Fig. 3m–p) and decreased CD31 (Fig. 3m and n) and VE-cadherin (Fig. 3o and p) expression.
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transfected with siRNA-CTRL did not exhibit any increase in either fibronectin (Fig. 2i and j) or collagen type III (Fig. 2k and l). Conversely, endothelial cells transfected with siRNA-TRPM4 showed an increase in fibronectin (Fig. 2i and j) and collagen type III (Fig. 2k and l) levels. These findings are in agreement with those obtained using the selective TRPM4 blocker 9-phenantrol (Supplementary Figure S3, http://links.lww.com/HJH/A458) [39].
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FIGURE 3 Cellular distribution of endothelial, fibrotic, and ECM proteins induced by the suppression of TRPM4 expression. ECs were transfected with a specific siRNA against the human TRPM4 isoform (siRNA-TRPM4) or a nontargeting siRNA [siRNA-control (CTRL)], and protein localization was detected. Representative images of the detection of the endothelial markers CD31 or VE-cadherin (red) and the fibrotic markers a-SMA, FSP-1, and FN (green). Boxes depicted in (a, c, e, g, i, k, m, and o) indicate the magnification shown in (b, d, f, h, j, l, n, and p), respectively. Arrows indicate CD31 (b, f, j, and n) or VE-cadherin (d, h, l, and p) labeling at the plasma membrane, whereas arrowheads indicate a-SMA (b and f), FSP-1 (d and h), or FN (j, n, l, and p) staining (n ¼ 5). Nuclei were stained using 4’-6-diamidino-2-phenylindole (DAPI). Scale bar represents 10 mm. ECs, endothelial cells; FM, fibronectin; siRNA, short interfering RNA; SMA, smooth muscle actin; TRPM4, transient receptor potential melastatin 4.
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findings, several profibrotic stimuli are supported by the production of TGF-b1 and TGF-b2. However, the association between TRPM4 expression and TGF-b isoform generation is currently unknown. Thus, we wondered whether the suppression of TRPM4 induces the production of TGF-b1 and TGF-b2 as a mechanism to promote endothelial fibrosis. Endothelial cells transfected with siRNA-TRPM4 showed increased TGF-b1 and TGF-b2 mRNA expression compared with cells transfected with siRNA-CTRL (Fig. 4a and b). Similarly, the downregulation of TRPM4 expression via siRNA-TRPM4 significantly increased the protein level of TGF-b1 and TGF-b2 in the supernatant (Fig. 4c–f) and in the protein extract (not shown). These data suggest that inhibiting TRPM4 expression promotes an increase in the expression and secretion of TGF-b1 and TGF-b2 in endothelial cells. Taking into account that TRPM4 downregulation induces the expression and secretion of TGF-b1 and TGF-b2, it is plausible that TGF-b isoforms are responsible for the increased [Ca2þ]i that is induced by inhibiting TRPM4 expression. Thus, we examined whether TGF-b plays a role in the calcium increases. To perform their actions, TGF-b isoforms activate ALK5 to induce gene transcription and promote fibrosis [40,41]. Thus, we performed our experiments using the specific ALK5 inhibitor SB431542. Typical doses of this ALK5 inhibitor are toxic to endothelial cell cultures. For this reason, we previously determined a lower, nontoxic yet still effective concentration of SB431542 to be used in our endothelial cell preparations [14]. Our results showed that endothelial cells transfected with siRNA-TRPM4 and incubated with several doses of SB431542 showed intracellular calcium increases similar to those observed in siRNA-TRPM4 treated endothelial cells in the absence of the ALK5 inhibitor (Fig. 4g). These results suggest the increase in the [Ca2þ]i induced by the inhibition in TRPM4 expression is largely independent of ALK5 activation.
To investigate whether the conversion of endothelial cells into activated fibroblast induced by TRPM4 suppression was mediated by TGF-b production, we tested whether its receptor, ALK5, is involved in the TRPM4 downregulation-induced endothelial fibrosis. Our data showed that endothelial cells transfected with siRNATRPM4 in the presence of SB431542 failed to decrease the levels of the endothelial marker VE-Cadherin (Fig. 5a and b). Furthermore, siRNA-TRPM4-treated endothelial cells exposed to SB431542 did not show any increase in the expression of the fibrotic proteins a-SMA (Fig. 5c and d) and fibronectin (Fig. 5e and f). These findings suggest that changes in endothelial and fibrotic markers observed on TRPM4-suppression-induced endothelial cell fibrosis are dependent on the ALK5 activity.
Suppressing transient receptor potential melastatin 4 induces the nuclear translocation of smad4 Upon the activation of ALK5 by TGF-b, smad2/3 proteins bind to smad4, inducing their migration to the nucleus to change protein expression and generate cell fibrosis [40,41]. Thus, smad4 translocation from the cytoplasm to the nucleus emerges as a crucial step in triggering and maintaining the fibrotic process. Given that TRPM4 downregulation promotes TGF-b1 and TGF-b2 production, we addressed the question of whether smad4 translocation is induced upon TRPM4 downregulation. In nontransfected endothelial cells (WT), smad4 was principally detected in the cytosol at every time observed (Fig. 6a–d). Similar results were observed with endothelial cells transfected with siRNA-CTRL (Fig. 6e–h). However, endothelial cells transfected with siRNA-TRPM4 showed a significant and clear translocation of smad4 to the nucleus (Fig. 6i–l), with smad4 being partially translocated at 8 and 12 h before transfection (Fig. 6j and k) and becoming almost completely translocated at 24 h post transfection (Fig. 6l).
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FIGURE 4 Production of TGF-b1 and TGF-b2 stimulated by TRPM4 downregulation in endothelial cells. (a and b) ECs were transfected with siRNA-control (CTRL) or siRNATRPM4, and TGF-b1(a) and TGF-b2(b) expression mRNA was analyzed. Results were normalized relative to 28S mRNA expression (n ¼ 6). (c–f) ECs were transfected with siRNA-CTRL or siRNA-TRPM4, and TGF-b1 (c and d) and TGF-b2 (e and f) protein expression was analyzed. (c and e) Representative images from western blot experiments performed to detect TGF-b1 (c) and TGF-b2 (e). (d and f) Densitometric analyses of the experiments shown in c and e, respectively. Protein levels were normalized against tubulin, and the data are expressed relative to siRNA-CTRL-transfected cells (n ¼ 6–9). Statistical significance was assessed using Student’s t-test (Mann–Whitney). P < 0.05. Graph bars show the mean SD. (g) Calcium overloading was evaluated using the Ca2þ-sensitive fluorescent dye Fluo-4 in nontransfected ECs [wild type (WT)] and in ECs transfected with siRNA-CTRL or siRNA-TRPM4 in the absence or presence of the ALK5-specific inhibitor SB431542 (n ¼ 5). Statistical significance was assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis) followed by Dunn’s post hoc test. P < 0.05; P < 0.01. Graph bars show the mean SD. ALK5, activin receptor-like kinase 5; ECs, endothelial cells; siRNA, short interfering RNA; TRPM4, transient receptor potential melastatin 4.
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FIGURE 5 TRPM4 downregulation-induced conversion of ECs into myofibroblasts is dependent on ALK5 activity. ECs were transfected with a specific siRNA against the human TRPM4 isoform (siRNA-TRPM4) or a nontargeting siRNA [siRNA-control (CTRL)], and protein expression was analyzed. (a–c). Representative images from western blot experiments performed to detect VE-cadherin (VE-Cad) (a), a-SMA (b), and FN (c). (d–f) Densitometric analyses of the experiments shown in a–c, respectively. Protein levels were normalized against tubulin, and the data are expressed relative to siRNA-CTRL-transfected cells (n ¼ 6). Statistical significance was assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis) followed by Dunn’s post hoc test. P < 0.05; P < 0.01. Graph bars show the mean SD. ALK5, activin receptor-like kinase 5; FM, fibronectin; siRNA, short interfering RNA; SMA, smooth muscle actin; TRPM4, transient receptor potential melastatin 4.
Densitometric analysis showed that the nucleus/cytosol ratio of smad4 was severely increased when endothelial cells were transfected with siRNA-TRPM4 (Fig. 6o), compared with nontransfected (WT) and siRNA-CTRL-transfected endothelial cells (Fig. 6m and n). The smad4 translocation observed upon siRNA-TRPM4 transfection was similar to that observed in endothelial cells exposed to TGF-b1 and TGF-b2, which was used as a positive control for smad4 translocation (supplemental Figure S4, http://links.lww.com/HJH/A458). In agreement with these results, protein extracts from the cytosol and nuclear fractions of nontransfected endothelial cells (WT) and cells transfected with siRNA-CTRL showed higher detection of smad4 in the cytosol fractions that in the nuclear fractions (Fig. 6p–s). In contrast, in endothelial cells transfected with siRNA-TRPM4, smad4 was observed predominantly in the nuclear fractions (Fig. 6p–s), suggesting that translocation was effectively stimulated by the suppression of TRPM4 expression.
DISCUSSION Endothelial dysfunction is a crucial part of systemic inflammation and affects endothelial cell monolayer integrity, which is essential for normal vascular function. Thus, the identification of relevant proteins that participate in the deleterious effects on endothelial cells is vital for improving current treatments. Here, we studied the role played by the TRPM4 ion channel, a regulator of intracellular Ca2þ levels, as a decisive factor in the conversion of endothelial cells into fibroblasts. In this study, we demonstrated that the suppression of TRPM4 expression increases intracellular Ca2þ levels. 988
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Inhibiting TRPM4 expression induces the conversion of endothelial cells into myofibroblasts. We found that the inhibition of TRPM4 expression increases fibrotic markers and decreases endothelial-specific proteins. Furthermore, TRPM4 downregulation increases the expression of ECM proteins. TRPM4 expression inhibition induces the expression and secretion of the endothelial fibrosis inducers TGFb1 and TGF-b2. Correspondingly, endothelial fibrosis induced by TRPM4 downregulation was dependent on ALK5 activity. In line with the development of the fibrotic process, intracellular Ca2þ levels were severely increased upon the suppression of TRPM4 expression independent of ALK5 activation. Consistently, TRPM4 downregulation induced the nuclear translocation of the profibrotic transcription factor smad4.
Mediators of inflammation play a role on endothelial fibrosis During acute and chronic systemic inflammation, proinflammatory cytokines circulate in the bloodstream and interact with the endothelial cells that form the inner wall of blood vessels. These inflammatory mediators maintain and enhance the inflammatory response through the activation of intracellular signaling in endothelial cells [6,7]. Several proinflammatory cytokines, including TGF-b, TNF-a, IL-6, and IL-1b, have been shown to be involved in the conversion of endothelial cells into activated fibroblasts through EndMT [8–12]. In addition, we demonstrated that the endotoxin lipopolysaccharide is also able to induce endothelial fibrosis [14]. Thus, the connection between endothelial fibrosis and mediators of inflammation has been well studied. Volume 33 Number 5 May 2015
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FIGURE 6 Suppression of TRPM4 induces the translocation of smad4 into the nucleus in endothelial cells. Smad4 was detected in nontransfected [wild type (WT)] (a–d) ECs and in ECs transfected with siRNA-control (CTRL) (e–h) or siRNA-TRPM4 (i–l) for 0, 8, 12, and 24 h. Representative images showing the detection of smad4 (green). Boxes depicted in (a, d, e, h, i, and l) indicate the magnification shown in (a0 , d0 , e0 , h0 , i0 , and l0 ), respectively. Nuclei were stained using 4’-6-diamidino-2-phenylindole (DAPI) Scale bar represents 30 mm. (m–o) Densitometric analyses of the experiments showed in d, h, and l, respectively. Data were expressed as the ratio of smad4 signal (pixels per mm2) in nucleus and cytosol (n ¼ 5). The numbers of analyzed cells are depicted in bars. Statistical significance was assessed using Student’s t-test (Mann– Whitney). P < 0.01. Graph bars show the mean SD. (p–s) Cytosol (CF)-enriched and nuclear (NF)-enriched fractions were obtained from nontransfected [wild type (WT)] ECs and from ECs transfected with siRNA-CTRL or siRNA-TRPM4, and smad4 expression was analyzed. (p–q) Representative images from western blot experiments performed to detect smad 4 in the CF and NF from nontransfected (WT, wild type) ECs and ECs transfected with siRNA-CTRL or siRNA-TRPM4. (r–s) Densitometric analyses of the experiments shown in p–q, respectively. Protein levels were normalized against tubulin or histone H3 for CF or NF, respectively, and the data are expressed relative to the nontransfected condition (n ¼ 5). Statistical significance was assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis test) followed by Dunn’s post hoc test. P < 0.01. Graph bars show the mean SD. ECs, endothelial cells; NS, nonsignificant; TRPM4, transient receptor potential melastatin 4.
Transient receptor potential melastatin 4 participates in Ca2þ dependent fibrotic features Changes in intracellular calcium levels are a decisive step in fibrosis development [15,16,20]. Recently, we showed that Journal of Hypertension
TRPM7 is a calcium channel that mediates endotoxininduced endothelial fibrosis [23]. To generate fibrosis, endotoxin challenge increases the intracellular calcium concentration in endothelial cells, which is dependent on TRPM7 activity [23]. Correspondingly, the inhibition of www.jhypertension.com
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TRPM7 expression inhibits endotoxin-induced endothelial fibrosis [23], suggesting that endotoxin-induced TRPM7mediated calcium influx is a crucial step in endothelial fibrosis. Furthermore, TRPM4 is involved in endothelial cell migration, which is a typical feature of activated fibroblasts that is mediated by intracellular calcium changes. It has been demonstrated that TRPM4 plays a decisive role in the enhancement of endothelial cell migration induced by oxidative stress [42,43].
Transient receptor potential melastatin 4 suppression-induced endothelial fibrosis as a potential mechanism for promoting endothelial dysfunction A relevant factor in the pathogenesis of acute and chronic systemic inflammation is the loss of endothelial function, otherwise known as endothelial dysfunction, such that normal endothelial functions are impaired, including deficient nitric oxide secretion, altered control of hemostasia, hyperpermeability, and cell death [44,45]. Broad endothelial dysfunction is observed during endotoxemiainduced systemic inflammation and is considered to be the cause of organ failure [44,46–48]. We reported that the endothelial death induced by endotoxin is inhibited by the suppression of TRPM4 activity and expression [30]. We showed that endothelial cells in which the activity or the expression of TRPM4 was decreased were resistant to endotoxin-induced cell death [30]. Interestingly, the morphology of those resistant endothelial cells, namely, cells not expressing TRPM4, showed a clear fibrotic-like phenotype [30], suggesting that these endothelial cells enter a fibrotic process as a means of escaping endotoxininduced death. These findings are in agreement with the data presented here, as endothelial cells transfected with siRNA-TRPM4 adopt a fibrotic-like phenotype in addition to acquiring several fibrotic markers and losing endothelial proteins. TRPM4 downregulation induces a change in the protein expression pattern of endothelial cells that promotes fibrotic-like protein expression similar to that previously reported in nontransfected wild-type endothelial cells exposed to TGF-b1 and TGF-b2 [8,9,49,50]. We observed that endothelial cells express and secrete both of these TGF-b isoforms when TRPM4 expression was suppressed. These results highlight a plausible mechanism for the induction endothelial cell fibrotic conversion upon TRPM4 downregulation. This is also supported by the observation that the TGF-b receptor ALK5 plays a crucial role in TRPM4 downregulation-induced endothelial fibrosis. Whether both TGF-b isoforms are equally responsible for the endothelial fibrosis remains an interesting issue, and further experiments are need to address this question. The mechanism underlying the production of TGF-b1 and TGF-b2 induced by inhibition of TRPM4 expression is not known. Launay et al. [32] demonstrated that the inhibition of TRPM4 expression increases the expression of IL-2 in T lymphocytes through the disruption of intracellular Ca2þ oscillations. Thus, in our model, TRPM4 downregulation may exert a similar effect. Further experiments are needed to determine whether the inhibition of TRPM4 expression 990
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enhances TGF-b expression via changes in intracellular Ca2þ oscillations. Intracellular signaling by TGF-b is mediated through binding to its plasma membrane receptor and the activation of smad2/3 proteins through phosphorylation. Once the TGF-b receptor promotes smad2/3 phosphorylation, smad4 is also bound and phosphorylated, thereby promoting its translocation to the nucleus to elicit gene transcription in support of the fibrotic process [40,41]. Thus, TRPM4 downregulation-induced smad4 nuclear translocation is consistent with the previous production of TGF-b, which stimulates smad4 translocation. In fact, the smad4 translocation induced by TRPM4 downregulation was similar to that observed using TGF-b1 and TGF-b2. Therefore, it is possible that TGF-b receptor inhibitors could significantly abolish the smad4 translocation induced by the inhibition of TRPM4 expression. Taken together, our results indicate that TRPM4 expression preserves the endothelial characteristics and that TRPM4 inhibition promotes fibrosis, which contributes to a mechanism to protect cells from pro-inflammatory cytokine-mediated endothelial death but also promotes myofibroblast production. Expressing TRPM4 at appropriate levels may be beneficial for the maintenance of endothelial phenotypes to avoid endothelial dysfunction during systemic or local inflammatory diseases.
ACKNOWLEDGEMENTS The authors are grateful to Director Dr Iva´nOyarzu´n and Dr Mario Carmona, Dr Jaime Mendoza and Mrs. Juana Belmar from Servicio de Ginecologı´a y Obstetricia, Hospital San Jose de Melipilla. Sponsorship: This work was supported by the research grants from FondoNacional de DesarrolloCientı´fico y Tecnolo´gico - Fondecyt 1121078 (F.S.), 3140448 (C.E.), 1120240 (D.V.), 1120286 (R.A.), and 1120380 (C.C.V.), Millennium Institute on Immunology and Immunotherapy P09-016-F (F.S.). Association-FrancaiseContre Les Myopathies AFM 16670 (C.C.V.), UNAB-DI-281-13/R (C.C.V.) and UNAB DI-209-12/N (F.S.).
Conflicts of interest There are no conflicts of interest.
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Reviewers’ Summary Evaluations
mation and endothelial dysfunction in disease models characterized by fibrosis.
Referee 1 Inflammation may promote endothelial dysfunction by converting endothelial cells into myofibroblasts. This study investigated whether transient receptor potential melastatin 4 (TRPM4) is involved in endothelial fibrosis, and to determine the mechanism. Using human endothelial cells, TRPM4 suppression using siRNA caused increased transforming growth factor-b expression, in association with increased intracellular calcium levels and expression of fibrotic and extracellular matrix markers, and decreased endothelial protein expression. Overall, these findings are consistent with TRPM4 regulating the endothelial phenotype, with reduced TRPM4 expression potentially promoting fibrosis. Further studies should investigate whether TRPM4 modulates inflam-
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Referee 2 Transient receptor potential melastatin 4 (TRPM4) channel has been identified as a key modulator of numerous calciumdependent physiological processes such as fibrosis. Although some calcium channels are known to be involved in fibrosis, the participation of TRPM4, a global regulator of intracellular calcium, in fibrosis development remains ambiguous. This study investigated the role of the TRPM4 in the endothelial-to-mesenchymal transition (EndMT), a possible mechanism for endothelial fibrosis. The authors found TRPM4 maintains endothelial features, and its loss promotes fibrotic conversion via TGF-b production. These results make the TRPM4 channel a promising novel target for treatment of EndMT-associated endothelial dysfunction
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