|Ahead of print publication
Dual inhibition by phosphodiesterase 5 and 5-HT2BInhibitor leads to near complete amelioration of fibrotic potential of human adult dermal fibroblasts isolated from a scleroderma patient
Saurabh Chaturvedi1, Mohit Kumar Rai1, Harshit Singh1, Durga Prasanna Misra1, Narayan Prasad2, Vinita Agrawal3, Vikas Agarwal1
1 Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Nephrology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Submission||20-Nov-2019|
|Date of Acceptance||19-May-2020|
Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Conversion of quiescent resident fibroblasts to activated myofibroblasts by transforming growth factor-beta 1 (TGF-β1) is the hallmark of fibrotic pathogenesis of systemic sclerosis (SSc). Myofibroblasts are characterized by increased alpha smooth muscle actin (α-SMA) expression and extracellular matrix (ECM) proteins production.
Objective: The aim of this study is to evaluate anti-fibrotic potential of dual inhibition by phosphodiesterase5 (PDE5) inhibitor, sildenafil plus 5-HT2Binhibitor, SB204741 in human adult dermal fibroblasts (HADFs) isolated from mid-forearm skin of a scleroderma patient.
Materials and Methods: In disease-mimicking strategy, HADFs were incubated with TGF-β1 (10 ng/ml) for 1 hour, followed by TGF-β1 (10 ng/ml)/[sildenafil (10 μM) or SB204741 (1 μM)] or combination of the two for 24 hours. In pretreatment strategy, HADFs were pretreated with sildenafil (10 μM) or SB204741 (1 μM) or combination of the two for 1 hour and later with only TGF-β1 (10 ng/ml) for 24 hours. Real time quantitative polymerase chain reaction for pro-fibrotic (COL1A1, COL1A2, ACTA2, CCN2 and FN1) and anti-fibrotic genes (MMP2, TIMP1) was performed.
Results: In TGF-β1 stimulated HADFs, upregulated expression of pro-fibrotic genes was observed (P < 0.05). Expression of pro-fibrotic genes in HADFs stimulated with TGF-β1 were significantly (P < 0.05) reduced by dual inhibition strategy with complete amelioration of ACTA2 in comparison to their respective individual treatments. Ratio of anti-fibrotic genes (MMP2/TIMP1) was restored significantly (P < 0.05).
Conclusion: Dual inhibition strategy with sildenafil plus SB204741 leads to near complete amelioration of conversion of fibroblasts to myofibroblasts and thus may have the potential to treat fibrosis in scleroderma.
Keywords: 5-HT2B receptor inhibitor, alpha smooth muscle actin, dermal fibroblasts, matrix mettaloproteinases, myofibroblasts, phosphodiesterase 5 inhibitor
|How to cite this URL:|
Chaturvedi S, Rai MK, Singh H, Misra DP, Prasad N, Agrawal V, Agarwal V. Dual inhibition by phosphodiesterase 5 and 5-HT2BInhibitor leads to near complete amelioration of fibrotic potential of human adult dermal fibroblasts isolated from a scleroderma patient. Indian J Rheumatol [Epub ahead of print] [cited 2021 Jan 27]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=298046
| Introduction|| |
Systemic sclerosis (scleroderma, SSc) is a connective tissue disease, which involves inflammation, vasculopathy, and fibrosis., The subsequent immuno-inflammation involved activates resident fibroblasts to myofibroblasts that secrete excessive extracellular matrix (ECM) proteins such as collagens and proteoglycans, which are thought to be key therapeutic targets., The above differentiation from resident fibroblasts to activated myofibroblasts is induced by pro-fibrotic growth factors such as transforming growth factor beta 1 (TGF-β1) or vasoactive peptide like serotonin (5-hydroxytryptamine, 5-HT).
TGF-β1 is the main pro-fibrotic factor that leads to fibrosis of the skin., Decreased blood flow and endovascular damage triggers and worsens skin fibrosis. Thus, targeting vasculopathy may lead to amelioration of fibrosis. Nitric oxide (NO) is endogenously synthesized from L-arginine through NO synthase., Vasodilation action of NO is mediated by downstream signaling molecules such as soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP),, which is a key mediator in NO/sGC/cGMP signaling, whose action is mediated by cGMP dependent protein kinase G (PKG) and cGMP gated channels. Cyclic nucleotide phosphodiesterases (PDEs) are a group of enzymes having at least 11 isoforms, which degrade the phosphodiester bond in cAMP and cGMP. Among 11 isoforms (PDE1-11), PDE5 is a cGMP-specific enzyme, which is abundant in vascular smooth muscle cells and has a strong vasodilatory effect. Sildenafil binds competitively to the active site of PDE5 and blocks its activity. Therefore, PDE5 inhibitors such as sildenafil and tadalafil have demonstrated brilliant outcomes for the treatment of SSc related pulmonary arterial hypertension (PAH) and digital ulcers.,,
In addition, recently, it has been observed that NO/sGC/cGMP signaling exerts anti-fibrotic activities,,, through stimulation of sGC, which thereby leads to increase in cGMP levels, resulting in reversing of fibrotic phenotype in SSc.,, Henceforth, PDE5 inhibitors have also been reported to have anti-fibrotic effects in models of various fibrotic disorders.
Outside the brain, serotonin, a vasoactive peptide, is synthesized and secreted by enterochromaffin cells of the gut. Platelets and mast cells uptake serotonin from the plasma via serotonin transporter. 5-HT is involved in the regulation of cell migration, proliferation, cytokine production, and vaso-regulation, exhibits well-characterized regulatory functions in multiple physiological systems. Seven families of 5-HT receptors mediate the cellular effects of 5-HT, (5-HT1 to 5-HT7). Most are G-protein coupled receptors, and 5-HT2A and 5-HT2B receptor subtypes have been shown to be involved in chronic fibrotic disorders such as retroperitoneal or pleural fibrosis, tissue fibrosis, carcinoid heart disease, and liver fibrosis.,, Recently, mitogenic and pro-fibrotic role of serotonin on different types of mesenchymal cells have been reported.
Since fibrosis can be mediated by multiple stimuli targeting different pathways, including TGF-β1, 5-HT, platelet-derived growth factor (PDGF), various mitogen-associated PKGs, it is likely that a mix of compounds targeting different pathways may be more effective in suppressing fibrotic pathways as compared to either alone. Therefore, we evaluated the efficacy of PDE5 inhibitor, sildenafil and, 5-HT2B receptor inhibitor, SB204741, individually as well as in combination for their anti-fibrotic efficacy.
| Materials and Methods|| |
Fibroblasts cell line
Human adult dermal fibroblasts (HADFs) were purchased from HIMEDIA, India (Lot no: 222342, product code: CL005, 42 years female, scleroderma patient) and maintained in their prescribed Dulbecco's Modified Eagle's medium (DMEM) under standard conditions [37°C/5% carbon dioxide (CO2)] added with heat-inactivated 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin-B. Cells were passaged by 0.25% trypsin-EDTA digestion and then washed and plated in DMEM. Cells between passages three to five were used for experiments., A detailed list of reagents used in this study are mentioned in supplementary materials 1.
TGF-ß1 standardization of dose
HADFs were stimulated with TGF-β1 at biologically relevant concentrations to evaluate the effect on pro-fibrotic genes expression., The detailed method is described in supplementary materials 2.
The effect of sildenafil and SB204741 on the proliferative capacity of HADFs was quantified using the reduction of MTT to insoluble purple formazan., The detailed method is described in supplementary materials 3.
Sildenafil and SB204741 standardization of dose
HADFs, in combination with TGF-β1 at biologically relevant concentrations, were treated with viable doses of Sildenafil and SB024741.,
Stimulation and antagonists treatments of HADFs
1 × 106 cells/well were seeded in 6 well plates for 24 hours. After adherence, HADFs were synchronized by incubating them with low serum medium (2% FBS in DMEM) for 24 hours. For stimulation of HADFs and treatment with inhibitors, sildenafil and SB204741, two strategies were adopted. In thefirst strategy, posttreatment strategy, HADFs were initially treated with TGF-β1 (10 ng/ml) for 1 hour and later incubated with TGF-β1 (10 ng/ml) and sildenafil or SB204741 (10 μM, 1 μM respectively) individually as well as in combination for 24 hours. In the second strategy, pretreatment strategy, HADFs were pretreated with sildenafil or SB204741 individually as well as in combination for 1 hour and later stimulated with only TGF-β1 (10 ng/ml) for 24 hours. Cells were stored, and RNA was extracted for performing real time polymerase chain reaction (PCR).
Quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR)
qRT-PCR was performed as described earlier. Detailed methodology is described in supplementary materials and methods 4. Primers for collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), smooth muscle alpha (α)-2 actin (ACTA2), connective tissue growth factor (CCN2 or CTGF) and fibronectin1 (FN1) and tissue inhibitor of metalloproteinase1 (TIMP1), matrix metalloproteinase2 (MMP2), TGF-β1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are listed in [Supplementary Table 1]. The expression of each target gene was normalized to GAPDH.
The results are represented as mean ± standard error of the mean (SEM). All the groups were evaluated for the level of significance using Student's t test. Significance was set at P ≤ 0.05. Data was analyzed and graphs were created using GraphPad prism software 6.0 program (GraphPad inc., CA, USA).
| Results|| |
Pro-fibrotic effects of TGF-ß1
TGF-β1 (2–20 ng/ml), respectively, induced pro-fibrotic genes mRNA expression in HADFs. The percentage viability of HADFs for TGF-β1 at 10 ng/ml was 80% [Figure Supplementary 1]a.
TGF-β1 dose-dependently induced the pro-fibrotic genes (COL1A1,COL1A2, and ACTA2) mRNA expression. For TGF-β1 maximal induction of pro-fibrotic genes mRNA was observed at a concentration of 10 ng/ml ([Figure 1], a-c). Higher concentrations of TGF-β1 did not further increase the expression of pro-fibrotic mRNA. Hence, TGF-β1 was associated with enhanced expression of pro-fibrotic genes in HADFs, supporting a potential pro-fibrotic role for TGF-β1 in mediating the pro-fibrotic phenotype of HADFs.
Pharmacological inhibition of phosphodiesterase5 and 5-HT2B receptor exerts anti-fibrotic effects in in vitro HADFs culture
To investigate whether sildenafil and SB204741 could suppress TGF-β1 induced fibrosis, the therapeutic potential of sildenafil and SB204741 was determined. The doses of sildenafil and SB204741 were chosen based on the published data., Of note, no evidence of the toxicity of anti-fibrotic doses of sildenafil (10 μM) and SB204741 (1 μM) were observed [Figure Supplementary 1]b and [Figure Supplementary 1]c).
Sildenafil and SB204741 decreased mRNA levels of COL1A1,COL1A2 and ACTA2 genes in a dose-dependent manner [Figure 2]a, [Figure 2]b, [Figure 2]c and [Figure 3]a, [Figure 3]b, [Figure 3]c respectively. Most prominent effects were observed at a concentration of 10 μM and 1 μM, respectively. Hence, a dose of 10 μM of sildenafil and 1 μM of SB204741 was chosen for further use.
|Figure 1: Effect of TGF-β1 on pro-fibrotic genes expression of HADFs: (a-c) mRNA levels of COL1A1 (a), COL1A2 (b), and ACTA2 (c) after stimulation with TGF-β1 of HADFs. Data was normalized with GAPDH as housekeeping gene Fold change in genes expression levels induced by different concentrations of TGF-β1 were compared to the HADFs cultured in media alone (blank). Results are shown as mean ± SEM of three independent experiments. Significance was determined by Student's t test. *P≤0.05, **P≤0.01 was considered as statistically significant as compared to blank. ns= non significant.|
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|Figure 2: Effect of various doses of sildenafil on pro-fibrotic genes mRNA expression stimulated with TGF-β1: mRNA levels of COL1A1, COL1A2 and ACTA2 after stimulation of HADFs with (a-c) TGF-β1 along with different doses of sildenafil (a-c). Data was normalized with GAPDH as housekeeping gene. Fold change in gene expression levels induced by different concentrations of (10 ng/ml) of TGF-β1 along with different concentrations of sildenafil were compared to the HADFs cultured in media alone (blank). Results are shown as mean ± SEM of three technical independent experiments. Significance was determined by Student's t test. *P≤0.05, **P≤0.01 was considered as statistically significant as compared to blank.|
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|Figure 3: Effect of various doses of SB204741 on pro-fibrotic genes mRNA expression stimulated with TGF-β1: mRNA levels of COL1A1, COL1A2 and ACTA2 after stimulation of HADFs with (a-c) TGF-β1 along with different doses of SB204741 (a-c). Data was normalized with GAPDH as housekeeping gene. Fold change in gene expression levels induced by different concentrations of (10ng/ml) of TGF-β1 along with different concentrations of SB204741 were compared to the HADFs cultured in media alone (blank). Results are shown as mean ± SEM of three independent experiments. Significance was determined by Student's t test. *P≤0.05, **P≤0.01 was considered as statistically significant as compared to blank.|
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Further, we assessed mRNA expression of pro-fibrotic genes: COL1A1,COL1A2,ACTA2,CCN2 and FN1. TGF-β1 at 10 ng/ml concentration upregulated the expressions of the COL1A1,COL1A2,ACTA2,CCN2, and FN1 mRNA at 24 hours [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e.
|Figure 4: Effect of sildenafil and SB204741 on TGF-β1 induced pro-fibrotic genes mRNA expression: HADFs (a-e) were cultured with only media (bar 1) and with TGF-β1 (bar 2). HADFs were pre-treated with TGF-β1 (10ng/ml) for 1 hour followed by co-culture with TGF-β1 (10ng/ml) and (10 μ M) dose of sildenafil (bar 3) and (1 μ M) dose of SB204741 (bar 4) respectively for 24 hours. HADFs were pre-treated with (10 μ M) dose of sildenafil (bar 5) and (1 μ M) dose of SB204741 (bar 6), respectively for 1 hour and later incubated with TGF-β1 (10ng/ml) only for 24 hours. mRNA levels of COL1A1 (a), COL1A2 (b), ACTA2 (c), CTGF (d), and FN1 (e) after above treatment strategies are depicted. Post-treatment strategy with (10μM) dose of sildenafil and (1μM) dose of SB204741 in combination (bar 7) and pre-treatment strategy with (10μM) dose of sildenafil and (1μM) dose of SB204741 in combination (bar 8) are shown. Data was normalized with GAPDH as housekeeping gene. Results are shown as mean ± SEM of three independent experiments. HADFs cultured only in the presence of media alone compared with TGF-β1 was represented as (#) and HADFs cultured in presence of sildenafil and SB204741 and TGF-β1 were represented as (##). Significance was determined by Student's t test*P≤0.05, **P≤0.01, ***P≤0.001 was considered as statistically significant.|
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Sildenafil and SB204741 significantly decreased the mRNA levels of COL1A1,COL1A2,ACTA2,CCN2 and FN1 in posttreatment strategy (disease mimicking strategy), [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e as well as in pretreatment strategy [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e individually and in combination. In pretreatment strategy, the dual combination of sildenafil and SB204741 almost nearly completely ameliorated ACTA2.
The expression of MMP2 and TIMP1 was affected by sildenafil but not by SB204741 [Figure 5]a, [Figure 5]b, [Figure 5]c. Sildenafil individually and in combination downregulated the expression of TIMP1 but surprisingly upregulated the expression of MMP2. Taken together, these data demonstrate that sildenafil and SB204741 mitigate the pro-fibrotic effects of TGF-β1. Sildenafil, however, also modulates anti-fibrotic genes expression.
|Figure 5: Effect of sildenafil and SB204741 on TGF-β1 induced anti-fibrotic genes mRNA expression: (a-c) HADFs were cultured with only media (bar 1) and with TGF-β1 (bar 2). HADFs were pre-treated with TGF-β1 (10 ng/ml) for 1 hour followed by co-culture with TGF-β1 (10 ng/ml) and 10 μ M dose of sildenafil (bar 3) and (1 μ M) dose of SB204741 (bar 4) respectively for 24 hours. HADFs were pre-treated with 10 μ M dose of sildenafil (bar 5) and (1 μ M) dose of SB204741 (bar 6) respectively for 1 hour and later incubated with TGF-β1 (10ng/ml) only for 24 hours. mRNA levels of TIMP1 (a), MMP2 (b) and MMP2/TIMP1 (c) after above treatment strategies are depicted. Post-treatment strategy with (10μM) dose of sildenafil and (1μM) dose of SB204741 in combination (bar 7) and pre-treatment strategy with (10μM) dose of sildenafil and (1μM) dose of SB204741 in combination (bar 8) are shown. Data was normalized with GAPDH as housekeeping gene. Results are shown as mean ± SEM of three independent experiments. HADFs cultured only in the presence of media alone compared with TGF-β1' (a-c) was represented as (#) and HADFs cultured in the presence of sildenafil and SB204741 and TGF-β1 (a-c) were represented as (##). Significance was determined by Student's t test. *P≤0.05, **P≤0.01, ***P≤0.001 was considered as statistically significant . ns= non significant.|
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| Discussion|| |
Inhibitors of PDE5 and 5-HT2B receptor, either alone or in combination, have a suppressive effect on TGF-β1 mediated pro-fibrotic effect on HADFs (fibroblasts derived from skin of scleroderma patient), however the combination of the two leads to near complete abrogation of conversion of quiescent fibroblasts to myofibroblasts.
Sildenafil is a PDE5 inhibitor, which is used for the treatment of PAH because of its strong vasodilation effects. Endothelial cells (ECs) dysfunction can be blamed for several vascular diseases, including SSc, in which ECs vascular tone impairment causes endothelin-1 (ET-1) overexpression and reduced NO production.,,,, In SSc, the earliest change is vasculopathy, which leads to ECs injury and subsequent platelet activation. Loss of anticoagulant properties of the endothelium may trigger the activation of platelets causing elevated plasma levels of 5-HT in SSc. The physiological levels of 5-HT are normally low as most 5-HT is pooled in the platelets, whereas in pathological conditions, 5-HT gets released from platelets and ECs, thereby increasing both locally and in the circulation., The fact that 5-HT has a role in fibrosis is evident from the histology of carcinoid tumors where fibrosis is evident both locally (peritumoral area) and systemically.
5-HT mediates its action directly via 5-HT2B receptor signaling or by conversion of latent TGF-β1 in plasma to active TGF-β1. In this study, 5-HT upregulated TGF-β1 mRNA expression in a dose-dependent manner, which we have reported earlier.
Further, in our opinion, the above experiments data show for thefirst time that sildenafil and SB204741 in combination in both treatment strategies; mimicking disease condition as well as pretreatment, could reverse fibrotic phenotype of the dermal fibroblasts more effectively than in individual treatment by interfering with pro-fibrotic effects of TGF-β1.
At the cellular level, basic etiology for fibrosis is supposed to be initiated by activation from a quiescent fibroblastic phenotype to a contractile myofibroblasts phenotype, which is characterized by the expression of α-SMA for which ACTA2 is the pro-fibrotic gene. When activated, myofibroblasts increase ECM synthesis and deposition, leading to the fibrotic changes characteristic of the onset of SSc. In this study, it has been demonstrated that in combination, sildenafil and SB204741 almost completely reduced the expression of ACTA2 at the mRNA level in HADFs after TGF-β1 stimulation. In addition, after incubation of HPFBs with TGF-β1, MMP2 is downregulated, and TIMP1 is regulated in comparison to blank. Therefore to eliminate the probability that induction of matrix degrading enzymes or their inhibitors suppress the decreased synthesis of ECM, expression of MMP2 and TIMP1 were analyzed in HADFs treated with TGF-β1, sildenafil, and SB204741. Sildenafil alone as well as in combination upregulated ratio of MMP2/TIMP1; however, SB204741 did not affect the expression of MMP2 and TIMP1.
| Conclusion|| |
The combination of, sildenafil and, SB204741, may be a novel treatment strategy for treating tissue fibrosis. In addition, our data provide further evidence that a combination of sildenafil and SB204741 has the potential to be an effective therapeutic strategy for fibrosis. The anti-fibrotic potency of sildenafil and SB204741 combination should be justified by further research on canonical and noncanonical signaling using invitro and animal models.
| Acknowledgement|| |
Mr. Saurabh Chaturvedi is a registered Ph.D. student from Dr. A.P.J. Abdul Kalam Technical University, Lucknow, Uttar Pradesh, India with registration number: Ph.D./13/PHARM/1502. We acknowledge the help and support from Dr. A.P.J. Abdul Kalam Technical University, Lucknow, Uttar Pradesh, India and also from Dr.K.C. Rastogi, Professor, Department of Pharmaceutics, Hygia Institute of Pharmaceutical Education and Research, Lucknow, Uttar Pradesh, India. Mr. Saurabh Chaturvedi was supported by a senior research fellowship grant from Indian Council of Medical Research, New Delhi, India (Grant No- 67/12/2014/IMM-BMS).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Higuchi T, Kawaguchi Y, Takagi K, Tochimoto A, Ota Y, Katsumata Y, et al
. Sildenafil attenuates the fibrotic phenotype of skin fibroblasts in patients with systemic sclerosis. Clin Immunol 2015;161:333-8.
Zhu X, Chu H, Jiang S, Liu Q, Liu L, Xue Y, et al
. Sirt1 ameliorates systemic sclerosis by targeting the mTOR pathway. J Dermatol Sci 2017;87:149-58.
Peng WJ, Yan JW, Wan YN, Wang BX, Tao JH, Yang GJ, et al
. Matrix metalloproteinases: A review of their structure and role in systemic sclerosis. J Clin Immunol 2012;32:1409-14.
Jinnin M. Mechanisms of skin fibrosis in systemic sclerosis. J Dermatol 2010;37:11-25.
Dees C, Akhmetshina A, Zerr P, Reich N, Palumbo K, Horn A, et al
. Platelet-derived serotonin links vascular disease and tissue fibrosis. J Exp Med 2011;208:961-72.
Gabrielli A, Avvedimento DV, Krieg T. Scleroderma. N
Eng J Med 2009;360:1989-2003.
Beyer C, Reich N, Schindler SC, Akhmetshina A, Dees C, Tomcik M, et al
. Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis. Ann Rheum Dis 2012;71:1019-26.
Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988;333:664-6.
Takimoto E. Cyclic GMP-dependent signaling in cardiac myocytes. Circ J 2012;76:1819-25.
Ramani GV, Park MH. Update on the clinical utility of sildenafil in the treatment of pulmonary arterial hypertension. Drug Des Devel Ther 2010;4:61-70.
Galiè N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D, et al
. Sildenafil citrate therapy for pulmonary arterial hypertension. N
Engl J Med 2005;353:2148-57.
Varga J, Pasche B. Transforming growth factor beta as a therapeutic target in systemic sclerosis. Nat Rev Rheumatol 2009;5:200-6.
Lin CS. Tissue expression, distribution, and regulation of PDE5. Int J Impot Res 2004;16 Suppl 1:S8-10.
Movsesian M, Stehlik J, Vandeput F, Bristow MR. Phosphodiesterase inhibition in heart failure. Heart Fail Rev 2009;14:255-63.
Maurice DH, Ke H, Ahmad F, Wang Y, Chung J, Manganiello VC. Advances in targeting cyclic nucleotide phosphodiesterases. Nat Rev Drug Discov 2014;13:290-314.
Cui W, Maimaitiyiming H, Qi X, Norman H, Zhou Q, Wang X, et al
. Increasing cGMP-dependent protein kinase activity attenuates unilateral ureteral obstruction-induced renal fibrosis. Am J Physiol Renal Physiol 2014;306:F996-1007.
Das A, Smolenski A, Lohmann SM, Kukreja RC. Cyclic GMP-dependent protein kinase Ialpha attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte. J Biol Chem 2006;281:38644-52.
Kass DA, Takimoto E. Regulation and role of myocyte cyclic GMP-dependent protein kinase-1. Proc Natl Acad Sci U S A 2010;107:E98.
Schinner E, Schramm A, Kees F, Hofmann F, Schlossmann J. The cyclic GMP-dependent protein kinase Iα suppresses kidney fibrosis. Kidney Int 2013;84:1198-206.
Geschka S, Kretschmer A, Sharkovska Y, Evgenov OV, Lawrenz B, Hucke A, et al
. Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats. PLoS One 2011;6:e21853.
Dunkern TR, Feurstein D, Rossi GA, Sabatini F, Hatzelmann A. Inhibition of TGF-beta induced lung fibroblast to myofibroblast conversion by phosphodiesterase inhibiting drugs and activators of soluble guanylyl cyclase. Eur J Pharmacol 2007;572:12-22.
Ferrini MG, Kovanecz I, Nolazco G, Rajfer J, Gonzalez-Cadavid NF. Effects of long-term vardenafil treatment on the development of fibrotic plaques in a rat model of Peyronie's disease. BJU Int 2006;97:625-33.
Mann DA, Oakley F. Serotonin paracrine signaling in tissue fibrosis. Biochim Biophys Acta 2013;1832:905-10.
Idzko M, Panther E, Stratz C, Müller T, Bayer H, Zissel G, et al
. The serotoninergic receptors of human dendritic cells: Identification and coupling to cytokine release. J Immunol 2004;172:6011-9.
Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 2002;71:533-54.
Grewal JS, Mukhin YV, Garnovskaya MN, Raymond JR, Greene EL. Serotonin 5-HT2A receptor induces TGF-beta1 expression in mesangial cells via ERK: Proliferative and fibrotic signals. Am J Physiol 1999;276:F922-30.
Königshoff M, Dumitrascu R, Udalov S, Amarie OV, Reiter R, Grimminger F, et al
. Increased expression of 5-hydroxytryptamine2A/B receptors in idiopathic pulmonary fibrosis: A rationale for therapeutic intervention. Thorax 2010;65:949-55.
Welsh DJ, Harnett M, MacLean M, Peacock AJ. Proliferation and signaling in fibroblasts: Role of 5-hydroxytryptamine2A receptor and transporter. Am J Respir Crit Care Med 2004;170:252-9.
Thwin MM, Douni E, Arjunan P, Kollias G, Kumar PV, Gopalakrishnakone P. Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1beta-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice. Arthritis Res Ther 2009;11:R138.
Chaturvedi S, Misra DP, Prasad N, Rastogi K, Singh H, Rai MK, et al
antagonists attenuate pro-fibrotic phenotype in human adult dermal fibroblasts by blocking TGF-β1 induced non-canonical signaling pathways including STAT3: Implications for fibrotic diseases like scleroderma. Int J Rheum Dis 2018;21:2128-38.
Ohkita M, Tawa M, Kitada K, Matsumura Y. Pathophysiological roles of endothelin receptors in cardiovascular diseases. J Pharmacol Sci 2012;119:302-13.
Chen Y, Leask A, Abraham DJ, Pala D, Shiwen X, Khan K, et al
. Heparan sulfate-dependent ERK activation contributes to the overexpression of fibrotic proteins and enhanced contraction by scleroderma fibroblasts. Arthritis Rheum 2008;58:577-85.
Ihn H, Yamane K, Tamaki K. Increased phosphorylation and activation of mitogen-activated protein kinase p38 in scleroderma fibroblasts. J Invest Dermatol 2005;125:247-55.
Chakraborty D, Šumová B, Mallano T, Chen CW, Distler A, Bergmann C, et al
. Activation of STAT3 integrates common profibrotic pathways to promote fibroblast activation and tissue fibrosis. Nat Commun 2017;8:1130.
Modlin IM, Shapiro MD, Kidd M. Carcinoid tumors and fibrosis: An association with no explanation. Am J Gastroenterol 2004;99:2466-78.
El-Tanbouly DM, Wadie W, Sayed RH. Modulation of TGF-β/Smad and ERK signaling pathways mediates the anti-fibrotic effect of mirtazapine in mice. Toxicol Appl Pharmacol 2017;329:224-30.
Hutcheson JD, Ryzhova LM, Setola V, Merryman WD. 5-HT2B antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation. J Mol Cell Cardiol 2012;53:707-14.
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