Anti-fibrotic effects of statin drugs: A review of evidence and mechanisms.

Fibrosis is a pathological repair process common among organs, that responds to tissue damage by replacement with non-functional connective tissue. Despite the widespread prevalence of tissue fibrosis, manifesting in numerous disease states across myriad organs, therapeutic modalities to prevent or alleviate fibrosis are severely lacking in quantity and efficacy. Alongside development of new drugs, repurposing of existing drugs may be a complementary strategy to elect anti-fibrotic compounds for pharmacologic treatment of tissue fibrosis. Drug repurposing can provide key advantages to de novo drug discovery, harnessing the benefits of previously elucidated mechanisms of action and already existing pharmacokinetic profiles. One class of drugs with a wealth of clinical data and extensively studied safety profiles is the statins, a class of antilipidemic drugs widely prescribed for hypercholesterolemia. In addition to these widely utilized lipid-lowering effects, increasing data from cellular, pre-clinical mammalian, and clinical human studies have also demonstrated that statins are able to alleviate tissue fibrosis originating from a variety of pathological insults via lesser-studied, pleiotropic effects of these drugs. Here we review literature demonstrating evidence for direct effects of statins antagonistic to fibrosis, as well as much of the available mechanistic data underlying these effects. A more complete understanding of the anti-fibrotic effects of statins may paint a clearer picture of their anti-fibrotic potential for various clinical indications. Additionally, more lucid comprehension of the mechanisms by which statins exert anti-fibrotic effects may aid in development of novel therapeutic agents that target similar pathways but with greater specificity or efficacy.

Comment on "Scar-Degrading Endothelial Cells as a Treatment for Advanced Liver Fibrosis".

Cellular therapies show promise for treatment of fibrosis. A recent article presents a strategy and proof-of-concept for delivering stimulated cells to degrade hepatic collagen in vivo. A discussion is presented surrounding the strengths of this approach and the potential to generalize this strategy of optimizing cell sources and activation stimuli to treat other types of fibrosis.

Epidermal Potentiation of Dermal Fibrosis: Lessons from Occlusion and Mucosal Healing.

Fibrotic skin conditions, such as hypertrophic and keloid scars, frequently result from injury to the skin and as sequelae to surgical procedures. The development of skin fibrosis may lead to patient discomfort, limitation in range of motion, and cosmetic disfigurement. Despite the frequency of skin fibrosis, treatments that seek to address the root causes of fibrosis are lacking. Much research into fibrotic pathophysiology has focused on dermal pathology, but less research has been performed to understand aberrations in fibrotic epidermis, leading to an incomplete understanding of dermal fibrosis. Herein, literature on occlusion, a treatment modality known to reduce dermal fibrosis, in part through accelerating wound healing and regulating aberrant epidermal inflammation that otherwise drives fibrosis in the dermis, is reviewed. The review focuses on epidermal-dermal crosstalk, which contributes to the development and maintenance of dermal fibrosis, an underemphasized interplay that may yield novel strategies for treatment if understood in more detail.

Prediction and Demonstration of Retinoic Acid Receptor Agonist Ch55 as an Antifibrotic Agent in the Dermis.

The prevalence of fibrotic diseases and the lack of pharmacologic modalities to effectively treat them impart particular importance to the discovery of novel antifibrotic therapies. The repurposing of drugs with existing mechanisms of action and/or clinical data is a promising approach for the treatment of fibrotic diseases. One paradigm that pervades all fibrotic diseases is the pathological myofibroblast, a collagen-secreting, contractile mesenchymal cell that is responsible for the deposition of fibrotic tissue. In this study, we use a gene expression paradigm characteristic of activated myofibroblasts in combination with the Connectivity Map to select compounds that are predicted to reverse the pathological gene expression signature associated with the myofibroblast and thus contain the potential for use as antifibrotic compounds. We tested a small list of these compounds in a first-pass screen, applying them to fibroblasts, and identified the retinoic acid receptor agonist Ch55 as a potential hit. Further investigation exhibited and elucidated the antifibrotic effects of Ch55 in vitro as well as showing antiscarring activity upon intradermal application in a preclinical rabbit ear hypertrophic scar model. We hope that similar predictions to uncover antiscarring compounds may yield further preclinical and ultimately clinical success.

Anti-fibrotic effects of pharmacologic FGF-2: a review of recent literature.

Fibrosis is a process of pathological tissue repair that replaces damaged, formerly functional tissue with a non-functional, collagen-rich scar. Complications of fibrotic pathologies, which can arise in numerous organs and from numerous conditions, result in nearly half of deaths in the developed world. Despite this, therapies that target fibrosis at its mechanistic roots are still notably lacking. The ubiquity of the occurrence of fibrosis in myriad organs emphasizes the fact that there are shared mechanisms underlying fibrotic conditions, which may serve as common therapeutic targets for multiple fibrotic diseases of varied organs. Thus, study of the basic science of fibrosis and of anti-fibrotic modalities is critical to therapeutic development and may have potential to translate across organs and disease states. Fibroblast growth factor 2 (FGF-2) is a broadly studied member of the fibroblast growth factors, a family of multipotent cytokines implicated in diverse cellular and tissue processes, which has previously been recognized for its anti-fibrotic potential. However, the mechanisms underlying this potential are not fully understood, nor is the potential for its use to ameliorate fibrosis in diverse pathologies and tissues. Presented here is a review of recent literature that sheds further light on these questions, with the hopes of inspiring further research into the mechanisms underlying the anti-fibrotic activities of FGF-2, as well as the disease conditions for which pharmacologic FGF-2 might be a useful option in the future.

Liposome-encapsulated statins reduce hypertrophic scarring through topical application.

Hypertrophic scar is an important clinical problem with limited therapeutic options. Aside from their roles as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins have also been demonstrated to decrease scarring by reducing connective tissue growth factor (CTGF) expression. However, poor penetrative ability limits their utility as topical treatments for hypertrophic scar. Here, we aim to develop novel statin formulations using liposomes to enhance dermal penetrative ability and to evaluate their efficacy against formation of hypertrophic scar utilizing our validated rabbit ear hypertrophic scar model. Liposomal simvastatin or pravastatin were compounded using a novel, flexible liposomal formulation and applied topically to rabbit ear hypertrophic scars daily from postoperation day (POD) 14 until POD 25. Scar color, including erythema and melanin, was measured using reflectance spectrophotometry on POD 28, and scar tissue was harvested for evaluation of scar elevation index as well as gene and protein expression. Human foreskin fibroblasts were also treated with statin formulations and CCN2 expression was determined by quantitative PCR. Both simvastatin and pravastatin were efficiently encapsulated in liposomes, forming nanometer-scale particles possessing highly negative charges. Topical treatment with liposomal simvastatin and pravastatin at 6.5% concentration significantly reduced scar elevation index and decreased type I/III collagen content and myofibroblast persistence in the wound. The erythema/vascularity of scars was reduced by liposomal statin treatment, with concomitant decrease of CD31 expression as measured histologically. Expression levels of transcripts encoding CTGF, collagen I, and collagen III collagen in scar tissue were also decreased by liposomal pravastatin treatment, as were myofibroblast persistence and the type I/III collagen ratio as assessed by immunofluorescence and picrosirus red staining, respectively. Treatment of human foreskin fibroblasts with simvastatin or with liposome-encapsulated pravastatin resulted in decreased expression of transcript encoding CTGF. Overall, our novel statin formulations encapsulated in liposomes were successfully delivered through topical application, significantly reducing hypertrophic scarring in a rabbit ear model.

Decellularized Fetal Matrix Suppresses Fibrotic Gene Expression and Promotes Myogenesis in a Rat Model of Volumetric Muscle Loss.

BACKGROUND: Traumatic muscle loss often results in poor functional restoration. Skeletal muscle injuries cannot be repaired without substantial fibrosis and loss of muscle function. Given its regenerative properties, the authors evaluated outcomes of fetal tissue-derived decellularized matrix for skeletal muscle regeneration. The authors hypothesized that fetal matrix would lead to enhanced myogenesis and suppress inflammation and fibrosis. METHODS: Composite tissue composed of dermis, subcutaneous tissue, and panniculus carnosus was harvested from the trunk of New Zealand White rabbit fetuses on gestational day 24 and from Sprague-Dawley rats on gestational day 18 and neonatal day 3, and decellularized using a sodium dodecyl sulfate-based negative-pressure protocol. Six, 10-mm-diameter, full-thickness rat latissimus dorsi wounds were created for each treatment, matrix was implanted (excluding the defect groups), and the wounds were allowed to heal for 60 days. Analyses were performed to characterize myogenesis, neovascularization, inflammation, and fibrosis at harvest. RESULTS: Significant myocyte ingrowth was visualized in both allogeneic and xenogeneic fetal matrix groups compared to neonatal and defect groups based on myosin heavy chain immunofluorescence staining. Microvascular networks were appreciated within all implanted matrices. At day 60, expression of Ccn2, Col1a1, and Ptgs2 were decreased in fetal matrix groups compared to defect. Neonatal matrix-implanted wounds failed to show decreased expression of Col1a1 or Ptgs2, and demonstrated increased expression of Tnf, but also demonstrated a significant reduction in Ccn2 expression. CONCLUSIONS: Initial studies of fetal matrices demonstrate promise for muscle regeneration in a rat latissimus dorsi model. Further research is necessary to evaluate fetal matrix for future translational use and better understand its effects.

Artesunate inhibits myofibroblast formation via induction of apoptosis and antagonism of pro-fibrotic gene expression in human dermal fibroblasts.

The anti-malaria drug artesunate and other chemical analogs of artemisinin have demonstrated cytostatic and cytotoxic effects in bacterial and cancer cells. Artemisinin-derived compounds have also been demonstrated to attenuate fibrosis in preclinical animal models, but the mechanisms by which this inhibition occurs are not well-understood. We investigated the effects of artesunate on the emergence of the myofibroblast, which is causally implicated in pro-fibrotic pathologies. CRL-2097 human dermal fibroblasts were analyzed for protein and transcript expression after treatment with artesunate to analyze fibroblast activation. Proliferation and apoptosis were also evaluated following treatment with artesunate in this cell line. Treatment of human dermal fibroblasts with artesunate antagonized fibroblast activation and pro-fibrotic extracellular matrix (ECM) deposition, both at basal culture conditions and when cultured in the presence of exogenous transforming growth factor-ß1 (TGF-ß1), a major pro-fibrotic cytokine. Artesunate-treated fibroblasts also demonstrated decreased proliferation and increased apoptosis. Transcript analysis by quantitative real-time polymerase chain reaction demonstrated that artesunate downregulated expression of pro-fibrotic genes including canonical myofibroblast markers, ECM genes, and several TGF-ß receptors and ligands, and upregulated expression of cell cycle inhibitors and matrix-metalloproteinases. Together, these data demonstrate that artesunate antagonizes fibroblast activation and decreases expression of pro-fibrotic genes, while also promoting myofibroblast apoptosis, suggesting that these mechanisms may be responsible in part for the anti-fibrotic effects of artesunate described previously.

Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-ß signaling results in variable activation of human dermal fibroblasts.

Fibroblast activation is a key step in the establishment of skin fibrosis induced by acute injury, and it is characterized by the differentiation of plastic resident tissue fibroblasts into contractile, extracellular matrix­secreting myofibroblasts. As fibroblast activation must be regulated in vivo, fibroblasts receive signals from the surrounding environment that initiate their fibrotic program. Thus, the present study investigated the effects of mitogen­activated protein kinase (MAPK) signaling pathways on fibroblast activation. It was demonstrated in primary human dermal fibroblasts that small molecule­mediated inhibition of extracellular signal­regulated kinase (ERK) and c­Jun N­terminal kinase (JNK) potentiated fibroblast activation, and that small molecule­mediated inhibition of p38 antagonized fibroblast activation. ERK and JNK inhibition cooperatively enhanced fibroblast activation mediated by treatment with exogenous transforming growth factor (TGF)­ß1, and p38 inhibition antagonized ERK inhibitor­mediated or JNK inhibitor­mediated fibroblast activation. Transcript analysis demonstrated that ERK and JNK inhibitor­mediated fibroblast activation was accompanied by distinct changes in the expression of TGF­ß­associated ligands and receptors, and that p38 inhibitor­mediated antagonism of fibroblast activation was accompanied by a distinct expression paradigm of TGF­ß­associated genes, including upregulation of betaglycan. ERK inhibitor­mediated and JNK inhibitor­mediated fibroblast activation was partially antagonized by small molecule­mediated inhibition of TGF­ß receptor (R)1, indicating that these mechanisms of fibroblast activation are partially dependent on TGF­ß/TGF­ßR signaling. These data collectively demonstrate and provide partial explanations of the varied effects and pathway dependencies of MAPK inhibitor­mediated effects on fibroblast activation.

Tryptophan metabolites kynurenine and serotonin regulate fibroblast activation and fibrosis.

Fibrosis is a pathological form of aberrant tissue repair, the complications of which account for nearly half of all deaths in the industrialized world. All tissues are susceptible to fibrosis under particular pathological sets of conditions. Though each type of fibrosis has characteristics and hallmarks specific to that particular condition, there appear to be common factors underlying fibrotic diseases. One of these ubiquitous factors is the paradigm of the activated myofibroblast in the promotion of fibrotic phenotypes. Recent research has implicated metabolic byproducts of the amino acid tryptophan, namely serotonin and kynurenines, in the pathology or potential pharmacologic therapy of fibrosis, in part through their effects on development of myofibroblast phenotypes. Here, we review literature underlying what is known mechanistically about the effects of these compounds and their respective pathways on fibrosis. Pharmacologic administration of kynurenine improves scarring outcomes in vivo likely not only through its well-characterized immunosuppressive properties but also via its demonstrated antagonism of fibroblast activation and of collagen deposition. In contrast, serotonin directly promotes activation of fibroblasts via activation of canonical TGF-ß signaling, and overstimulation with serotonin leads to fibrotic outcomes in vivo. Recently discovered feedback inhibition between serotonin and kynurenine pathways also reveals more information about the cellular physiology of tryptophan metabolism and may also underlie possible paradigms for anti-fibrotic therapy. Together, understanding of the effects of tryptophan metabolism on modulation of fibrosis may lead to the development of new therapeutic avenues for treatment through exploitation of these effects.

Fibroblast Growth Factor 2 as an Antifibrotic: Antagonism of Myofibroblast Differentiation and Suppression of Pro-Fibrotic Gene Expression.

Fibrosis is a pathological condition that is characterized by the replacement of dead or damaged tissue with a nonfunctional, mechanically aberrant scar, and fibrotic pathologies account for nearly half of all deaths worldwide. The causes of fibrosis differ somewhat from tissue to tissue and pathology to pathology, but in general some of the cellular and molecular mechanisms remain constant regardless of the specific pathology in question. One of the common mechanisms underlying fibroses is the paradigm of the activated fibroblast, termed the "myofibroblast," a differentiated mesenchymal cell with demonstrated contractile activity and a high rate of collagen deposition. Fibroblast growth factor 2 (FGF2), one of the members of the mammalian fibroblast growth factor family, is a cytokine with demonstrated antifibrotic activity in non-human animal, human, and in vitro models. FGF2 is highly pleiotropic and its receptors are present on many different cell types throughout the body, lending a great deal of variety to the potential mechanisms of FGF2 effects on fibrosis. However, recent reports demonstrate that a substantial contribution to the antifibrotic effects of FGF2 comes from the inhibitory effects of FGF2 on connective tissue fibroblasts, activated myofibroblasts, and myofibroblast progenitors. FGF2 demonstrates effects antagonistic towards fibroblast activation and towards mesenchymal transition of potential myofibroblast-forming cells, as well as promotes a gene expression paradigm more reminiscent of regenerative healing, such as that which occurs in the fetal wound healing response, than fibrotic resolution. With a better understanding of the mechanisms by which FGF2 alters the wound healing cascade and results in a shift away from scar formation and towards functional tissue regeneration, we may be able to further address the critical need of therapy for varied fibrotic pathologies across myriad tissue types.

FGF2-mediated attenuation of myofibroblast activation is modulated by distinct MAPK signaling pathways in human dermal fibroblasts.

BACKGROUND: Previous human and animal studies have demonstrated the ability of exogenously administered basic fibroblast growth factor (FGF2) to act as an antifibrotic agent in the skin. Though the activity of FGF2 as an anti-scarring agent is well-established for fibrotic skin wounds, the mechanisms by which FGF2 exerts these actions are not entirely understood. Canonical FGF2 signaling proceeds in part via FGFR/MAPK pathways in human dermal fibroblasts, and FGF2 has been described to prevent or reverse the fibroblast-to-myofibroblast transition, which is driven by TGFß signaling and understood to be an important step in the formation of a fibrotic scar in vivo. Thus, we set out to investigate the antagonistic effects of FGF2 on TGFß signaling as well as the broader effects of MAPK inhibition on the TGFß-mediated induction of myofibroblast gene expression. OBJECTIVE: To better understand the effects of FGF2 signaling pathways on myofibroblastic gene expression and cell phenotypes. METHODS: Human dermal fibroblasts were cultured in vitro in the presence of FGF2, TGFß, and/or MAPK inhibitors, and the effects of these agents were investigated by molecular biology techniques including qRT-PCR, immunofluorescence, Western blot, and flow cytometry. RESULTS: FGF2 inhibited TGFß-mediated fibroblast activation, resulting in more rapidly proliferating, spindle-shaped cells, compared to the more slowly proliferating, flatter TGFß-treated cells. Treatment with FGF2 also attenuated TGFß-mediated increase in expression of myofibroblast markers smooth muscle α-actin, calponin, transgelin, connective tissue growth factor, ED-A fibronectin, and collagen I. FGF2-mediated antagonism of the TGFß-mediated fibroblast-to-myofibroblast transition was reversed by small molecule inhibition of ERK or JNK, and it was potentiated by inhibition of p38. MAPK inhibition was demonstrated to have qualitatively similar effects even in the absence of exogenous FGF2, and small molecule inhibition of p38 MAPK was sufficient to attenuate TGFß-mediated fibroblast activation. CONCLUSIONS: Inhibition of select MAPK signaling pathways can reverse or potentiate anti-fibrotic FGF2 effects on human dermal fibroblasts, as well as exert their effects independently of exogenous FGF2 supplementation.

PRMT8 demonstrates variant-specific expression in cancer cells and correlates with patient survival in breast, ovarian and gastric cancer.

Recent emphasis has been placed on the role of epigenetic regulators and epigenetic marks as biomarkers for cancer diagnosis and prognosis, and as therapeutic targets for treatment. One such class of regulators is the protein arginine methyltransferase (PRMT) family. The present study examined available curated data regarding the expression and alteration of one of the least studied PRMT family members, PRMT8, in various types of cancer and cancer cell lines. Publicly available cancer data on PRMT8 expression were examined using the Human Protein Atlas and the Kaplan-Meier Plotter, and reverse transcription-polymerase chain reaction was used to screen a selection of human cell lines for variant-specific PRMT8 expression. High levels of PRMT8 expression in breast, ovarian and cervical cancer was observed. Additionally, in patients with breast and ovarian cancer, high PRMT8 expression was correlated with increased patient survival, whereas in gastric cancer, high PRMT8 expression was correlated with decreased patient survival. The present study also investigated the expression of PRMT8 variant 2, a novel transcript variant recently identified in our laboratory, in various cancer cell lines. Variant-specific expression of PRMT8 in numerous distinct cancer cell lines derived from different tissues, including the expression of the novel PRMT8 variant 2 in U87MG glioblastoma cells was demonstrated. The present study proposes the possibility of PRMT8 as a cancer biomarker, based on the high level of PRMT8 expression in various types of cancer, particularly in tissues that would not normally be expected to express PRMT8, and on the correlation of PRMT8 and patient lifespan in several cancer types. Variant-specific expression of PRMT8 in diverse cancer cell lines suggests the possibility of alternate PRMT8 isoforms to have diverse effects on cancer cell phenotypes.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass.