Liver fibrosis is a common feature and end-stage condition of many chronic liver diseases with various etiologies. As the main complication of chronic liver damage, liver fibrosis is essentially a wound healing process characterized by the accumulation of ECM proteins in the liver [12,13] that leads to scarring and impairment of liver function. Several studies found that RA may play a protective role against ECM accumulation and liver fibrosis in vivo. Wang L et al.[14] found that ATRA reduced the amount of histological detectable fibrosis induced by carbon tetrachloride (CCl₄) in C57BL/6J mice, and this was accompanied by an attenuation of the accumulation of collagen α2 (I). That ATRA ameliorates CCl₄-induced liver fibrosis was confirmed by Hisamori et al.[15] in BALB/c mice. Equally, Yang KL et al.[16] reported that a RA derivative isolated from the mycelium of Phellinus linteus could antagonize ECM accumulation and liver fibrosis in BALB/c mice by down-regulating reactive oxygen species (ROS) generation and calcium influx, thereby directly affecting transforming growth factor-β1 (TGF-β1). Wang H and co-workers [17] showed that ATRA (1.5 and 7.5 mg/kg) was able to inhibit common bile duct ligation (CBDL)-induced liver fibrosis in female Wistar rats, and ATRA reduced the expression of collagen I protein more greatly than that of 0.1 mg/kg. The expression of type I collagen (COL-I), tissue inhibitors of metalloproteinase-1 (TIMP-1), TGF-β1, and connective tissue growth factor (CTGF) were reduced, whilst He H et al.[18] reported that 5 mg/kg ATRA significantly reduced liver fibrosis and nearly eliminated liver necrosis after CBDL in male Sprague-Dawley rats, especially in combination with ursodeoxycholic acid (UDCA). Most importantly, they found that ATRA alone or in combination with UDCA repressed CYP7A1 expression in human hepatocytes, and significantly inhibited collagen 1A1 (COL-1A1), matrix metalloproteinase-2 (MMP-2), and α-smooth muscle actin (α-SMA) expression and/or activity in primary human hepatic stellate cells (HSCs) and LX-2 cells (a HSC cell line), and TGF-β1 induced Smad2 phosphorylation in LX-2 cells.

In vitro, a number of studies also found that RA could play a protective role against ECM accumulation. For instance, Hellemans et al.[19] reported that ATRA exerted a significant inhibitory effect on the synthesis of procollagen I, III, and IV, FN, and LN in HSCs. Conversely, 9-cis-RA increased procollagen I, but did not affect the expression of other matrix proteins. Recently, Ye Y et al.[20] reported that ATRA inhibits proliferation and collagen production in HSCs via attenuation of the mRNA expression of collagene genes, i.e., procollagen α1 (I) and α1 (III), and the profibrogenic genes TGF-β1, CTGF, MMP-2, TIMP-1, TIMP-2, and plasminogen activator inhibitor-1 (PAI-1). Concurrently, the mRNA expression of MMP-3 and MMP-13 was stimulated by suppression of c-Jun N-terminal kinase (JNK) and active protein-1 (AP-1). The notion that RA and its derivatives could prevent ECM accumulation in HSCs was further corroborated by Yang K et al.[16] and He H and collaborators [18]. The latter group reported that 5 μM ATRA significantly suppressed COL-1A1 mRNA expression by more than 50% in HSCs [18]. Hisamori S et al.[15] performed a study in HSCs and disclosed that the administration of ATRA down-regulated the production of TGF-β1, interleukin-6 (IL-6), collagen, nuclear factor-κB p65, and p38 mitogen activated protein kinase (p38MAPK). Radaeva et al.[21] showed that in early activated HSCs compared with quiescent or fully activated HSCs, RA and retinal dehydrogenase (Raldh) levels were upregulated. They also showed that blocking RA synthesis with a Raldh inhibitor or a retinoic acid receptor antagonist abolished the up-regulation of retinoic acid early inducible gene 1 (RAE-1), which in a chronic status would lead to chronic liver fibrosis. Conversely, treatment with RA or oxidation of retinol to RA induced RAE-1 expression in HSCs, and sensitizes early activated HSCs to NK cell killing.

Despite the aforementioned results, three reports suggest that quite the opposite occurs and that RA enhances ECM production and exacerbates liver fibrosis both in vitro and in vivo. Interestingly, those three reports from the same study group and thus lack independent confirmation. Okuno et al.[22] reported that 9-cis-RA exacerbated rat liver fibrosis by inducing the activation of TGF-β1 in male Wistar rats. However, the used dose in their study was markedly higher compared with the doses used in other in vivo studies (40 mg/kg of body weight, 5 times a week; see Table 2). They also performed a study in vitro using HSCs, and found that RA enhanced plasminogen activator (PA)/plasmin levels and thereby induced proteolytic activation of TGF-β1, a strong fibrogenic cytokine, resulting in enhanced ECM production [22,23].

In agreement with the majority of studies mentioned above, Ye et al. found that a lower dose of RA could inhibit the expression of TGF-β1 and could play a protective role against cell injury, proliferation, and collagen accumulation [20]. However, at higher doses, Okuno showed that RA up-regulated the expression of TGF-β1 and negatively influenced ECM accumulation and liver fibrosis [22]. There might be a common pathway in which RA displays a protective effect or an adverse effect, which is obviously dose dependent.

The characteristics of the studies mentioned above in which the effect of RA on liver fibrosis were evaluated are summarized in Table 2, and we found that the used dose of RA in the study by Okuno et al.[22] was significantly higher than in other studies, especially in the animal experiments.

Pulmonary fibrosis is an intractable paranchymal lung disease that is characterized by persistent alveolitis and accumulation of connective tissue and ECM [24]. The pathogenesis of pulmonary fibrosis is not well understood, but the occurrence of fibrotic lesions is often due to secondary disease causes or irritants, e.g., viral infections, sarcoidosis, systemic sclerosis, etc., long-term exposure to occupational and/or environmental inhalants, radiation from cancer treatment, certain drugs, or idiopathic in origin. The currently available in vitro and in vivo reports indicate that RA plays a protective role and prevents ECM accumulation in the lung, thereby protecting against pulmonary fibrosis. In C57BL/6 female mice, Tabata et al.[25] demonstrated that ATRA prevented radiation- or bleomycin-induced pulmonary fibrosis. They found that ATRA reduced irradiation-induced IL-6 production through the protein kinase C (PKC)-δ/NF-κB pathway, and inhibited irradiation-induced TGF-β1 production through the p38MAPK/NF-κB pathway, resulting in the inhibition of cell differentiation and collagen synthesis. Esteban-Pretel et al.[26] found that RA could reduce the amount of collagen IV and down-regulate the expression of proinflammatory cytokines, interleukin-1α (IL-1α), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in vitamin A-deficient rats. However, they also recorded that RA treatment increased oxidative stress and lipid peroxidation, most likely because of auto-oxidation of RA due to the high pO₂ in the lung that produced superoxide and carbon-centered radicals.

Ozer et al.[27] reported that the degree of fibrosis and α-SMA expression showed a significant decrease in RA-treated newborn Wistar rats with oxygen-induced lung injury. Tabata et al.[28] found that ATRA could reduce IL-6 expression and concomitantly NF-κB, blocked IL-6R and gp130, and likely reduced radiation-induced pulmonary fibrosis via a PKCδ-dependent pathway. Dong Z and co-workers [29] recently showed that ATRA is able to decrease the expression of interleukin-17A (IL-17A), IL-6, and TGF-β1, and could alleviate bleomycin-induced pulmonary fibrosis in C57BL/6 male mice. In vitro, Tabata C et al.[25] found that ATRA reduced irradiation-induced production of IL-6, TGF-β1, α-SMA and COL-1A1 in lung fibroblasts. Tabata C and collaborators [28] also found that ATRA reduced the activated form of NF-κB p65 and reduced the expression of IL-6 in lung fibroblasts.

All studies examined, report positive effects on pulmonary fibrosis under treatment with RA. The characteristics of the studies mentioned above evaluating the effect of RA on pulmonary fibrosis are summarized in Table 3.

Kidney fibrosis is a major and final pathological manifestation of progressive kidney disease and is characterized by the activation of fibroblasts, epithelial-to-mesenchymal transition, monocyte and/or macrophage infiltration, and excessive ECM accumulation along with a loss of functioning nephrons [30]. This contributes to a progressive decrease in glomerular filtration and tubular function. Numerous studies performed in cellulo and animal models report that ATRA is able to alleviate glomerulonephritis and renal interstitium disease.

From in vivo studies, Wagner et al.[31] reported that both ATRA and 13-cis-RA significantly reduced glomerular α-SMA and alleviated glomerular proliferation, glomerular lesions, and albuminuria in a Thy1.1-induced mesangioproliferative glomerulonephritis rat model. In continued investigations by the same group, Morath et al.[32] went on to show that the beneficial effects of the retinoids, in particular ATRA, on glomerular damage could be attributed to a distinct reduction in renal TGF-β1 and TGF receptor II (TGFRII) expression. Lehrke et al.[33] reported that treatment of glomerulonephritic rats with agonists specific for the retinoid X receptor (Ro-257386) completely inhibited induction of TGF-β1, procollagen I, and FN expression in Thy1.1-induced glomerulonephritis rats. In turn, Oseto et al.[34] found that ATRA also significantly reduced proliferating cell nuclear antigen (PCNA), ED-1, α-SMA, TNF-α, IL-1β, and CCAAT enhancer-binding protein 8 (C/EBP8) in glomerulonephritis rats. Furthermore, down-regulation of the cell proliferation-related platelet derived growth factor (PDGF) and fibrosis-related TGF-β1, and COL-1 were recorded. Schaier et al.[35] reported that both the retinoic acid receptor alpha (RARα-specific agonist AGN 195183 and the retinoid X receptor (RXR) specific agonist AGN 194204 reduced the gene expression of glomerular TGF-β1 and prepro-ET1, and alleviated renal damage in rats with established chronic glomerulonephritis (chronic mesangioproliferative Thy-GN (MoAb 1-22-3) rat model). Adams et al.[36] found that 13-cis-RA acted as a potent immunosuppressive and antifibrotic agent able to prevent and inhibit the progression of glomerulosclerosis and interstitial fibrosis in a chronic Fisher344→Lewis kidney allograft rat model. The mRNA expression of the chemokines monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2), macrophage inflammatory protein-1α/chemokine (C-C motif) ligand 3 (MIP-1α/CCL3), interferon gamma-induced protein 10/C-X-C motif chemokine 10 (IP-10/CXCL10), and Regulated And Normal T cell Expressed and Secreted/chemokine (C-C motif) ligand 5 (RANTES/CCL5), and proteins associated with fibrosis, i.e., PAI-1, TGF-β1, and collagens I and III, were strikingly lower in treated allografts. Recently, He et al.[37] established that ATRA inhibited cell proliferation and induces differentiation in HIV-infected podocytes through RARα-mediated cAMP/PKA activation, whilst in HIV-1-transgenic mice they recorded reduced proteinuria, cell proliferation, and glomerulosclerosis. Xu et al.[38] performed a highly interesting study using three doses of ATRA (low doses, 6–10.7 mg/kg/day; medium doses, 12.7–18.8 mg/kg/day; high doses, 20.1–27.4 mg/kg/day) in Alb/TGF-β1 transgenic mice (induces progressive renal fibrosis and retinoic acid deficiency in the kidneys), and reported that the low doses had a tendency to reduce the average urinary albumin excretion, as well as renal retinaldehyde dehydrogenase 2 (RALDH2; catalyzes the synthesis of RA from retinaldehyde), FN, COL-1A1, COL-1A2 and COL-4A1 compared with the control group (C57BL/6J × CBA F1 mice). Nonetheless, the extent of renal fibrosis remained unchanged. Most importantly, with increasing dose, reduction of the aforementioned factors disappeared, mortality rates increased, and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased. This study underscores the Janus-like properties of RA, and therefore further studies are desperately needed to determine where RA’s profibrotic and toxic action begins and ends. In vitro, Adams et al.[36] performed a study in peritoneal macrophages, and found that 13-cis-RA could pronouncedly decrease the protein secretion of inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-10).

In human mesangial cells, Wen et al.[39] reported that 9-cis-RA attenuated TGF-β1-induced α-SMA, FN, and PAI-1 expression, but it did not significantly affect cell proliferation and survival. As the authors express it, it is interesting that part of their study shows that 9-cis-RA induced hepatocyte growth factor (HGF) mRNA expression and protein secretion, stimulated HGF promoter activity, activated c-met receptor phosphorylation, and induced expression of the Smad transcriptional co-repressor TGIF. In an overexpression approach of TGIF or treatment with 9-cis-RA resulted in suppression of trans-activation of the TGF-β-responsive promoter and conditional ablation of the c-met receptor completely abolished the anti-fibrotic effect of 9-cis-RA and abrogated TGIF induction. Collectively, the results of these studies suggest that 9-cis-RA exerts its anti-fibrotic action by antagonizing TGF-β1 and that this is possibly mediated via a HGF/c-met receptor signaling mechanism. Liu and co-workers [40] found that treatment with two different doses of ATRA (5 mg/kg/day and 10 mg/kg/day by gastric gavage) lowed the glomerulosclerosis index and increased renal function in 5/6 nephrectomized Sprague Dawley rats, which was at least partly attributable to a reduction in PAI-1 and α-SMA expression; plasmin and MMP-2 levels remained unchanged. Kishimoto et al.[41] equally showed that ATRA could significantly improve the histological and immunological overall image, including the extent of macrophage infiltration and could improve the expression of MCP-1, TGF-β1, α-SMA and collagen I in unilaterally ureterally obstructed C57BL/B6 mice. Importantly, the authors determined in preliminary experiments that ATRA induced apoptosis, predominantly in the infiltrating cells, which might be an important reason for the improvement in renal interstitial fibrosis. Mallipattu et al.[42] found that ATRA could up-regulate the gene expression of GSTA4, PER2, IFRD1, HIVEP1, LIPE, METTL1, RFX1, SNX5, PLAUR, CLCN3, SFXN4, DMD, FOXC1, KLF15, GDNF, GABARAPL1 and could down-regulate the gene expressions of GATA3, CCND2, ISGF3G, MCAM, SIAT1, FGF18, PITX2, GNB4, WISP1, YWHAZ, CACNA1C in cultured podocytes. Our previous studies [10,43,44] distinctly showed that ATRA was able to increase the expression of MMP-2 and MMP-9, and reduce the expression of apoE, Col-IV, FN, and TGF-β1 in glomerulosclerosis rats. And we also found in renal interstitial fibrosis rats that the expression of prohibitin was increased by ATRA (15 mg/kg/day).

However, despite the numerous reports that demonstrate positive effects on renal fibrosis, a few studies reported that ATRA treatment exacerbated the glomerulonephritis and renal interstitium disease. For instance, Iyoda et al.[45] found that the renal pathology in cryoglobulin-associated membranoproliferative glomerulonephritis mice (mice overexpressing thymic stromal lymphopoietin [TSLP]) was aggravated after treatment with ATRA (20 mg/kg 3 times weekly by intraperitoneal injection), and significantly increased glomerular collagen IV in cryoglobulin-associated membranoproliferative glomerulonephritis mice at 4 weeks but not 8 weeks. Beside these changes, the authors reported systemic deterioration and unexpected changes in immunoglobulins. They propose that the negative effects induced by ATRA might be an immunomodulatory effect of ATRA on B cell function. Moulder et al.[46] reported that ATRA exacerbated radiation nephropathy in rats and they speculated that this negative effect might be the result of stimulation of renal cell proliferation or inhibition of renal nitric oxide activity. Likewise, Alique and coworkers [47] demonstrated that ATRA exacerbated glycated albumin effects on intracellular oxidation and the expression of the molecules involved in leucocyte infiltration in cultured human mesangial cells. In patients treated with ATRA for acute promyelocytic leukemia, retinoic acid syndrome (RAS), which is characterized by an inflammatory reaction with capillary leakage and myeloid cell tissue invasion that presents with cardiopulmonary symptoms and occasionally acute kidney injury, is a serious and documented complication [48]. Such studies suggest that caution should be exercised when translating ATRA treatment of human disease based on the largely positive animal data reported in the literature.

The characteristics of the studies that evaluated the effect of RA on kidney fibrosis discussed here are summarized in Table 4.

The role that RA plays in fibrosis of various other tissues and organs, and the signaling pathways involved are equally diverse as in the aforementioned organs. It becomes clear that not only signaling with respect to constituents of the ECM, but also mediators or inhibitors of inflammation, oxidative stress and others are involved.

Choudhary et al.[49] reported that ATRA could significantly reduce interstitial and perivascular fibrosis in aortic restricted Sprague-Dawley rats. RA inhibited the cleavage of caspase-3 and-9, restored the ratio of Bcl-2 to Bax, and prevented a decrease in SOD-1 and SOD-2 levels in pressure-overloaded rats. Furthermore, the authors state that pressure overload-induced phosphorylation of ERK1/2, JNK, and p38 was inhibited by RA via up-regulation of mitogen-activated protein kinase phosphatase 1 and 2 (MKP-1; MKP-2). The pressure overload-induced production of angiotensin II was attenuated by RA via up-regulation of angiotensin-converting enzyme 2 (ACE 2) and through the inhibition of the expression of cardiac and renal renin, angiotensinogen, ACE1, and angiotensin type 1 receptor (AGT1R). Klopcic and co-workers [50] found that ATRA reduced inflammation, tissue destruction, and fibrosis in a 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced intestinal fibrosis mice model, whilst indomethacin increased TNBS-induced fibrosis. The authors also suggest that the reported opposing effects may be linked via secreted protein acidic and rich in cysteine (SPARC); an acidic ECM glycoprotein that plays an important role in cell-matrix interactions, collagen binding, and bone mineralization.

Okoshi et al.’s [51] research shows that administration of ATRA attenuated irradiation-induced intestinal fibrosis and concomitantly reduced the expression of IL-6 and TGF-β1. They could determine from further in vitro studies that ATRA suppressed the trans-differentiation of irradiated intestinal fibroblasts and reduced the production of α-SMA and collagen. Wang et al.[52] reported that ATRA could reduce the expressions of TGF-β1 and collagen I in peritoneum tissue and could prevent peritoneal fibrosis in rat model of peritoneal dialysis. Treharne et al.[53] found that RA could reduce the extent of the fibrotic lesions in cystic fibrosis, and could increase the expression of tissue transglutaminase (Tgase2); Tgase2 crosslinks proteins between ɛ-amino groups a lysine residues and γ-carboxamide groups of glutamine residues (increased stability and resistance to proteolysis) and also acts as a G-protein. In turn, Tgase2 induced the expression of nucleoside diphosphate kinase (NDPK), a membrane-bound protein kinase that catalyzes the exchange of phosphate groups between different nucleosides and is known to control G-protein function.

The signaling pathways for RA in miscellaneous fibrosis are summarized in Figure 1.