Myofibroblast Formation Research Articles

Case Report: Corneal Inlay Removal Following Myofibroblast Detection under in Vivo Confocal Microscopy.

Corneal inlays, one of the treatment options for presbyopia, are effective and safe, and various such devices have been developed. However, there have been cases of inlay removal due to complications or patient dissatisfaction. The purpose of this study was to report a case of inlay removal due to corneal opacity after inlay implantation, and the results of 5 years of follow-up. A 63-year-old man was referred to our hospital with visual disturbance and double vision in his left eye. Two years prior to presentation at our hospital, he had undergone bilateral laser in situ keratomileusis with corneal inlay implantation in the left eye at another clinic. Slit-lamp examinations showed paracentral corneal opacity. The patient was treated with tranilast eye drops for 18 months, with no progression of symptoms. However, six months after stopping the eye drop treatment, the opacity recurred and vision acuity decreased, along with the formation of myofibroblasts around the inlay, as revealed by in vivo confocal microscopy. Consequently, the inlay was removed at the previous clinic. During the subsequent five-year follow-up period, ophthalmic examination revealed reduced corneal opacity, although visual acuity did not change; moreover, no myofibroblast was found. Corneal inlays can sometimes cause complications. In this case, the patient experienced corneal fibrosis and associated vision loss. In vivo confocal microscopy detected myofibroblasts that cause corneal stromal fibrosis; thus, the removal was decided to avoid fibrosis progression.

Exosomes from tubular epithelial cells undergoing epithelial-to-mesenchymal transition promote renal fibrosis by M1 macrophage activation.

Kidney fibrosis is the common final pathway of chronic kidney disease (CKD), and it is distinguished by inflammation, mesenchymal transition with myofibroblast formation, and epithelial-to-mesenchymal transition (EMT). Macrophages are protuberant inflammatory cells in the kidney, and their roles are dependent on their phenotypes. However, it remains unclear whether tubular epithelial cells (TECs) undergoing EMT can influence the phenotypes of macrophages and the underlying mechanisms during the development of kidney fibrosis. Here, we investigated the characteristics of TECs and macrophages during kidney fibrosis with a focus on EMT and inflammation. We found that the coculture of exosomes from transforming growth factor-beta (TGF-β)-induced TECs with macrophages induced macrophage M1 polarization, while exosomes from TECs without TGF-β stimulation or stimulation with TGF-β alone did not induce an increase in M1 macrophage-related markers. Notably, TECs induced to undergo EMT by TGF-β treatment released more exosomes than the other groups. Furthermore, it is noteworthy that when we injected exosomes from TECs undergoing EMT into mice, in addition to the high level of inflammatory response and the activation of M1 macrophages, the indicators of EMT and renal fibrosis in mouse kidney tissue were correspondingly elevated. In summary, exosomes from TECs undergoing EMT by TGF-β treatment induced M1 polarization and led to a positive feedback effect for further EMT and the development of renal fibrosis. Therefore, the obstacle to the release of such exosomes may be a novel therapeutic strategy for CKD.

Modulating Myofibroblastic Differentiation of Fibroblasts through Actin-MRTF Signaling Axis by Micropatterned Surfaces for Suppressed Implant-Induced Fibrosis.

Myofibroblasts, the primary effector cells for implant-induced fibrosis, contribute to this process by secreting excessive collagen-rich matrix and contracting. Thus, approaches that suppress myofibroblasts may achieve desirable suppression effects in the fibrotic process. As one of the important physical properties of materials, material topographical structures have been proven to affect various aspects of cell behaviors, so is it possible to manipulate the formation of myofibroblasts by tailoring the topographical properties of medical devices? In this study, polycaprolactone (PCL) surfaces with typical micropatterns (micro column and micro pit) were fabricated. The regulatory effects of surface micropatterns on the myofibroblastic differentiation of fibroblasts were investigated. Compared to the flat surfaces and surfaces with micro pit, surfaces with micro columns triggered the F- to G-actin transition, inhibiting the nuclear transfer of myocardin-related transcription factor-A. Subsequently, the downstream gene α-smooth muscle actin, which is a marker of myofibroblasts, was suppressed. Further in vivo investigation showed that PCL implants with micro-column-patterned surfaces inhibited the formation of peri-implant fibrotic capsules. Our results demonstrate that surface topographical properties are a potent regulator of fibroblast differentiation into myofibroblasts and highlight the antifibrotic potential of modifying surfaces with micro-column patterns.

Diminished schwann cell repair responses play a role in delayed diabetes-associated wound healing.

Diabetes mellitus is the most common metabolic disease associated with impaired wound healing. Recently, Schwann cells (SCs), the glia of the peripheral nervous system, have been suggested to accelerate normal skin wound healing. However, the roles of SCs in diabetic wound healing are not fully understood. In this study, Full-thickness wounds were made in the dorsal skin of C57/B6 mice and db/db (diabetic) mice. Tissue samples were collected at different time points, and immunohistochemical and immunofluorescence analyses were performed to detect markers of de-differentiated SCs, including myelin basic protein, Sox 10, p75, c-Jun, and Ki67. In addition, in vitro experiments were performed using rat SC (RSC96) and murine fibroblast (L929) cell lines to examine the effects of high glucose conditions (50mM) on the de-differentiation of SCs and the paracrine effects of SCs on myofibroblast formation. Here, we found that, compared with that in normal mice, wound healing was delayed and SCs failed to rapidly activate a repair program after skin wound injury in diabetic mice. Furthermore, we found that SCs from diabetic mice displayed functional impairments in cell de-differentiation, cell-cycle re-entry, and cell migration. In vitro, hyperglycemia impaired RSC 96 cell de-differentiation, cell-cycle re-entry, and cell migration, as well as their paracrine effects on myofibroblast formation, including the secretion of TGF-β and Timp1. These results suggest that delayed wound healing in diabetes is due in part to a diminished SC repair response and attenuated paracrine effects on myofibroblast formation.

MALAT1-miR-30c-5p-CTGF/ATG5 axis regulates silica-induced experimental silicosis by mediating EMT in alveolar epithelial cells

Epithelial-mesenchymal transdifferentiation of alveolar type Ⅱ epithelial cells is a vital source of pulmonary myofibroblasts, and myofibroblasts formation is recognized as an important phase in the pathological process of silicosis. miR-30c-5p has been determined to be relevant in the activation of the epithelial-mesenchymal transition (EMT) in numerous disease processes. However, elucidating the role played by miR-30c-5p in the silicosis-associated EMT process remains a great challenge. In this work, based on the establishment of mouse silicosis and A549 cells EMT models, miR-30c-5p was interfered with in vivo and in vitro models to reveal its effects on EMT and autophagy. Moreover, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), connective tissue growth factor (CTGF), autophagy-related gene 5 (ATG5), and autophagy were further interfered with in the A549 cells models to uncover the possible molecular mechanism through which miR-30c-5p inhibits silicosis associated EMT. The results demonstrated the targeted binding of miR-30c-5p to CTGF, ATG5, and MALAT1, and showed that miR-30c-5p could prevent EMT in lung epithelial cells by acting on CTGF and ATG5-associated autophagy, thereby inhibiting the silicosis fibrosis process. Furthermore, we also found that lncRNA MALAT1 might competitively absorb miR-30c-5p and affect the EMT of lung epithelial cells. In a word, interfering with miR-30c-5p and its related molecules (MALAT1, CTGF, and ATG5-associated autophagy) may provide a reference point for the application of silicosis intervention-related targets.

Hyponatremia-related liver steatofibrosis and impaired spermatogenesis: evidence from a mouse model of the syndrome of inappropriate antidiuresis

PurposeHyponatremia is the most frequent electrolytic disorder in clinical practice. In addition to neurological symptoms, hyponatremia, even when mild/moderate and chronic, has been related to other manifestations, such as bone demineralization and increased risk of fractures. To better elucidate tissue alterations associated with reduced serum sodium concentration [Na+], we developed an in vivo model of hyponatremia secondary to the Syndrome of Inappropriate Antidiuresis.Methods and resultsHyponatremia was induced in Foxn1nu/nu mice by subcutaneous infusion of the vasopressin analog 1-deamino [8-D-arginine] vasopressin (dDAVP) for 14 days via osmotic mini-pumps. Mice in the control group were infused with isotonic saline solution. Serum [Na+] progressively decreased, with a nadir of 123.4 ± 2.3 mEq/L (mean ± SD, dDAVP 0.3 ng/h) and 111.6 ± 4.7 mEq/L (mean ± SD, dDAVP 0.5 ng/h). Evident signs of liver steatofibrosis were observed at histology in hyponatremic mice. Accordingly, the expression of proteins involved in lipid metabolism (SREBP-1, PPARα and PPARγ) and in myofibroblast formation (αSMA and CTGF) significantly increased. Furthermore, heme oxygenase 1 expression was up-regulated in Kupffer and hepatic stellate cells in the liver of hyponatremic mice. Testis alterations were also observed. In particular, the thickness of the seminiferous epithelium appeared reduced. The expression levels of PCNA and PTMA, which are involved in DNA replication and germ cells maturation, were markedly reduced in the testis of hyponatremic mice.ConclusionOverall, these findings shed new light on the possible consequences of chronic hyponatremia and prompt a more thorough evaluation of hyponatremic patients.

Statins synergize with other drugs to prevent myofibroblast transformation in in vitro model of peyronie's disease

Peyronie's disease is characterized by formation of a plaque in the tunica albuginea (TA), with myofibroblasts being the main effector cells. We have previously shown a synergy between phosphodiesterase type 5 inhibitors (PDE5is) and selective estrogen receptor modulators (SERMs) in inhibiting myofibroblast formation in models of PD. Further research revealed statins to be anti-fibrotic as well. Whilst the anti-fibrotic effect of statins themselves has already been described, we aimed to combine statins with our previously identified drugs. Tunica albuginea (TA) of PD patients was used to isolate primary fibroblasts. TA-derived fibroblasts were exposed to Transforming Growth Factor-ß1 (TGF-β1) and a range of concentrations of statins, PDE5i, SERM, and their combinations for 72h before quantifying α-smooth muscle actin (α-SMA) using In-Cell ELISA (ICE). Statins (simvastatin, lovastatin) showed a concentration dependent anti-fibrotic effect to prevent TGF-ß1-induced myofibroblast transformation, with IC50 values of 0.77 µM and 0.8 µM for simvastatin and lovastatin, respectively. Addition of 0.3 µM of simvastatin to a full concentration response curve of vardenafil revealed drug synergy, as 0.3 µM of simvastatin showed 12% inhibition, 0.3 µM of vardenafil 1.5% inhibition, but their combination exerted 33% inhibition. Similarly, 0.18 µM of tamoxifen showed 2% inhibition, whilst a combination with 0.3 µM simvastatin resulted in 24% inhibition. Interestingly, combining simvastatin, vardenafil, and tamoxifen was not more effective than vardenafil and tamoxifen, or vardenafil and simvastatin only. Statins, PDE5i, and SERMs are anti-fibrotic, and both double combinations (statin-PDE5i, statin-SERM) showed synergy in vitro. A triple combination did not exceed the double combinations. However, care must be taken with attempts to translate these results to bedside, as there have been reports of strong adverse effects in patients, such as myopathy due to metabolic toxicity, when treated with a statin-PDE5i combination. Authors hold patent for PDE5i-SERM use in fibrosis

Abstracts from the NIH Office of Research on Women's Health 2022 Annual BIRCWH Meeting:Building Interdisciplinary Research Careers in Women's Health November 2, 2022

Abstracts from the NIH Office of Research on Women's Health 2022 Annual BIRCWH Meeting:Building Interdisciplinary Research Careers in Women's Health November 2, 2022

Upregulation of ppET-1/ETBR/eNOS mRNA Expression After Calcitriol Treatment in Chronic Kidney Diseases Model in Rats

Introduction: Myofibroblast formation in the interstitial area is the hallmark of chronic kidney disease (CKD). Endothelin signalling has been known to play role in physiology and pathophysiology in the kidney. Vitamin D has a reno-protective effect through inhibiting inflammation and fibrosis. However, the interaction between vitamin D and endothelin signalling in the CKD model has not been elucidated yet. Therefore, we aimed to check the difference impact of endothelin (ET) receptor in CKD. Methods: Sprague Dawley rats (3-months-old, 150-250grams) underwent 5/6 subtotal nephrectomy (SN) to induce CKD. Then, it was divided into 4 groups (each contains 6 rats): sham operation (SO), 5/6 subtotal nephrectomy (SN), calcitriol groups (0.01µg/100grBW/day (SN-D1), and 0.05µg/100grBW/day (SN-D2). Calcitriol was administered for 14 days after the surgery. The Sham Operation (SO) group was injected with NaCl. At the specified date, the rats were sacrificed and the kidneys were harvested. Fibrosis was quantified based on Sirius Red staining. Immunostaining was done for localizing fibroblast (PDGFRβ). The mRNA expressions of prepro-ET-1, endothelin receptor A (ETAR), endothelin receptor B (ETBR), and endothelial nitrite oxide synthase (eNOS) were quantified using reverse-transcriptase PCR (RT-PCR). Results: The CKD promotes an elevation of prepro-ET-1, ETBR, and eNOS, and reduction of ETAR (p<0.05) mRNA expression compared to the SO group. Administration of calcitriol (SN-D1 and SN-D2) showed the vice versa effects. However, only SN-D2 group consistently showed statistically significant differences whenever compared to either SO or SN groups. Conclusion: Calcitriol might attenuate interstitial fibrosis in CKD model via ET-1/eNOS signalling.

Jmjd3/IRF4 axis aggravates myeloid fibroblast activation and m2 macrophage to myofibroblast transition in renal fibrosis.

Renal fibrosis commonly occurs in the process of chronic kidney diseases. Here, we explored the role of Jumonji domain containing 3 (Jmjd3)/interferon regulatory factor 4 (IRF4) axis in activation of myeloid fibroblasts and transition of M2 macrophages into myofibroblasts transition (M2MMT) in kidney fibrosis. In mice, Jmjd3 and IRF4 were highly induced in interstitial cells of kidneys with folic acid or obstructive injury. Jmjd3 deletion in myeloid cells or Jmjd3 inhibitor reduced the levels of IRF4 in injured kidneys. Myeloid Jmjd3 depletion impaired bone marrow-derived fibroblasts activation and M2MMT in folic acid or obstructive nephropathy, resulting in reduction of extracellular matrix (ECM) proteins expression, myofibroblasts formation and renal fibrosis progression. Pharmacological inhibition of Jmjd3 also prevented myeloid fibroblasts activation, M2MMT, and kidney fibrosis development in folic acid nephropathy. Furthermore, IRF4 disruption inhibited myeloid myofibroblasts accumulation, M2MMT, ECM proteins accumulation, and showed milder fibrotic response in obstructed kidneys. Bone marrow transplantation experiment showed that wild-type mice received IRF4-/- bone marrow cells presented less myeloid fibroblasts activation in injured kidneys and exhibited much less kidney fibrosis after unilateral ureteral obstruction. Myeloid Jmjd3 deletion or Jmjd3 inhibitor attenuated expressions of IRF4, α-smooth muscle actin and fibronectin and impeded M2MMT in cultured monocytes exposed to IL-4. Conversely, overexpression IRF4 abrogated the effect of myeloid Jmjd3 deletion on M2MMT. Thus, Jmjd3/IRF4 signaling has a crucial role in myeloid fibroblasts activation, M2 macrophages to myofibroblasts transition, extracellular matrix protein deposition, and kidney fibrosis progression.

Role of MMP3 and fibroblast-MMP14 in skin homeostasis and repair

Early lethality of mice with complete deletion of the matrix metalloproteinase MMP14 emphasized the proteases’ pleiotropic functions. MMP14 deletion in adult dermal fibroblasts (MMP14Sf-/-) caused collagen type I accumulation and upregulation of MMP3 expression. To identify the compensatory role of MMP3, mice were generated with MMP3 deletion in addition to MMP14 loss in fibroblasts. These double deficient mice displayed a fibrotic phenotype in skin and tendons as detected in MMP14Sf-/- mice, but no additional obvious defects were detected. However, challenging the mice with full thickness excision wounds resulted in delayed closure of early wounds in the double deficient mice compared to wildtype and MMP14 single knockout controls. Over time wounds closed and epidermal integrity was restored. Interestingly, on day seven, post-wounding myofibroblast density was lower in the wounds of all knockout than in controls, they were higher on day 14. The delayed resolution of myofibroblasts from the granulation tissue is paralleled by reduced apoptosis of these cells, although proliferation of myofibroblasts is induced in the double deficient mice. Further analysis showed comparable TGFβ1 and TGFβR1 expression among all genotypes. In addition, in vitro, fibroblasts lacking MMP3 and MMP14 retained their ability to differentiate into myofibroblasts in response to TGFβ1 treatment and mechanical stress. However, in vivo, p-Smad2 was reduced in myofibroblasts at day 5 post-wounding, in double, but most significant in single knockout, indicating their involvement in TGFβ1 activation. Thus, although MMP3 does not compensate for the lack of fibroblast-MMP14 in tissue homeostasis, simultaneous deletion of both proteases in fibroblasts delays wound closure during skin repair. Notably, single and double deficiency of these proteases modulates myofibroblast formation and resolution in wounds.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence

BackgroundFibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. MethodsStable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1−/−) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. ResultsCarbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFβ-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. ConclusionsThese results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Abstract P2050: Acetyl-coa Synthesis Is Required For Chromatin Remodeling In Myofibroblast Differentiation And Persistence

Heart failure (HF) features fibrotic remodeling by myofibroblasts, a differentiated fibroblast population responsible for wound repair. Discovering mechanisms of myofibroblast formation and function may yield novel targets to delay or reverse HF. Myofibroblasts metabolically reprogram to mediate transcriptional activation of fibrotic genes through chromatin remodeling. We previously reported that α-ketoglutarate production for histone demethylation is essential for cardiac fibrosis, but metabolic regulation of histone acetylation in fibrosis is unclear. ATP-citrate lyase (ACLY) and Acetyl-coenzyme A synthetase 2 (ACSS2) synthesize acetyl-CoA in the cytoplasm and have also been reported to supply substrate for histone acetyltransferases in the nucleus to modify regulatory histone residues. To investigate acetyl-CoA metabolism in myofibroblast formation, we stimulated adult mouse and human cardiac fibroblasts (CFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with pharmacological inhibitors of ACLY and ACSS2. Either ACLY or ACSS2 inhibition sufficiently reverted differentiated myofibroblasts to a non-fibrotic phenotype, even during continuous TGFβ stimulation. Genetic deletion of ACLY in fibroblasts recapitulated these results. Further, ACLY inactivation was sufficient to decrease global histone acetylation. ACLY deletion, specifically in activated myofibroblasts in the adult mouse heart, slowed LV functional decline and remodeling in a model of pressure overload HF. Our data indicate that ACLY and ACSS2 are necessary for myofibroblast differentiation and persistence. We hypothesize that acetyl-CoA synthesis is necessary for histone acetylation required for activation of myofibroblast genes. Ongoing work will determine the molecular mechanisms of both enzymes in chromatin remodeling and in cardiac injury. In summary, acetyl-CoA producing enzymes are attractive clinical targets to reverse cardiac fibrosis.

Pharmacological Inhibition of STING/TBK1 Signaling Attenuates Myeloid Fibroblast Activation and Macrophage to Myofibroblast Transition in Renal Fibrosis.

Renal fibrosis is an important pathological biomarker of chronic kidney disease (CKD). Stimulator of interferon genes/TANK binding kinase 1 (STING/TBK1) axis has been identified as the main regulator of innate immune response and closely related to fibrotic disorder. However, the role of STING/TBK1 signaling pathway in kidney fibrosis is still unknown. In this study, we investigated the effect of pharmacological inhibition of STING/TBK1 signaling on renal fibrosis induced by folic acid (FA). In mice, TBK1 was significantly activated in interstitial cells of FA-injured kidneys, which was markedly inhibited by H-151 (a STING inhibitor) treatment. Specifically, pharmacological inhibition of STING impaired bone marrow-derived fibroblasts activation and macrophage to myofibroblast transition in folic acid nephropathy, leading to reduction of extracellular matrix proteins expression, myofibroblasts formation and development of renal fibrosis. Furthermore, pharmacological inhibition of TBK1 by GSK8612 reduced myeloid myofibroblasts accumulation and impeded macrophage to myofibroblast differentiation, resulting in less deposition of extracellular matrix protein and less severe fibrotic lesion in FA-injured kidneys. In cultured mouse bone marrow-derived monocytes, TGF-β1 activated STING/TBK1 signaling. This was abolished by STING or TBK1 inhibitor administration. In addition, GSK8612 treatment decreased levels of α-smooth muscle actin and extracellular matrix proteins and prevents bone marrow-derived macrophages to myofibroblasts transition in vitro. Collectively, our results revealed that STING/TBK1 signaling has a critical role in bone marrow-derived fibroblast activation, macrophages to myofibroblasts transition, and kidney fibrosis progression.

Sinensetin protects against pulmonary fibrosis via inhibiting Wnt/β-Catenin signaling pathway

Pulmonary fibrosis is a fatal lung disease characterized by progressive fibroblast proliferation and extensive extracellular matrix (ECM) deposition. Although great efforts have been placed to understand the pathogenesis and explore therapeutic strategies, the treatment options for pulmonary fibrosis are very limited and the prognosis of pulmonary fibrosis patients remains poor. Sinensetin is a polymethoxylated flavone derived from citrus fruits, and has been characterized with anti-fibrotic property. However, the underlying mechanism is still unclear. In present study, combining in vitro and in vivo models, we revealed for the first time that sinensetin can protect against pulmonary fibrosis via inhibiting glycogen synthase kinase-3β (GSK-3β)-mediated Wnt/β-Catenin signaling pathway. According to our results, the activation of Wnt/β-Catenin signaling pathway in pulmonary fibrosis is responsible for fibroblast proliferation and differentiation to form ECM-producing myofibroblasts. Sinensetin treatment dephosphorylates and activates GSK-3β, a component of β-Catenin destruction complex, which induces β-Catenin degradation and deactivates Wnt/β-Catenin-mediated fibroblast proliferation and differentiation. As a result, myofibroblasts formation and ECM production are reduced and the progression of pulmonary fibrosis is suppressed. These results not only advance our knowledge on the pharmacological activities of sinensetin, but also provide novel insights on pulmonary fibrosis treatment.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts invitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit invivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n= 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p<0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p<0.001), fibronectin (3.2-fold; p<0.001), collagen I (0.8-fold; p<0.001), and collagen III (1.4-fold; p<0.001), as well as protein levels of α-SMA (23.8%; p<0.001) and collagens (1.8-fold; p<0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-β signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Histone deacetylase 3 promotes alveolar epithelial–mesenchymal transition and fibroblast migration under hypoxic conditions

Epithelial–mesenchymal transition (EMT), a process by which epithelial cells undergo a phenotypic conversion that leads to myofibroblast formation, plays a crucial role in the progression of idiopathic pulmonary fibrosis (IPF). Recently, it was revealed that hypoxia promotes alveolar EMT and that histone deacetylases (HDACs) are abnormally overexpressed in the lung tissues of IPF patients. In this study, we showed that HDAC3 regulated alveolar EMT markers via the AKT pathway during hypoxia and that inhibition of HDAC3 expression by small interfering RNA (siRNA) decreased the migration ability and invasiveness of diseased human lung fibroblasts. Furthermore, we found that HDAC3 enhanced the migratory and invasive properties of fibroblasts by positively affecting the EMT process, which in turn was affected by the increased and decreased levels of microRNA (miR)-224 and Forkhead Box A1 (FOXA1), respectively. Lastly, we found this mechanism to be valid in an in vivo system; HDAC3 siRNA administration inhibited bleomycin-induced pulmonary fibrosis in mice. Thus, it is reasonable to suggest that HDAC3 may accelerate pulmonary fibrosis progression under hypoxic conditions by enhancing EMT in alveolar cells through the regulation of miR-224 and FOXA1. This entire process, we believe, offers a novel therapeutic approach for pulmonary fibrosis.

Oral Progenitor Cell Line-Derived Small Extracellular Vesicles as a Treatment for Preferential Wound Healing Outcome.

Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell–mediated repair in varied settings; hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow–derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings.

Fibroblast growth factor 2 inhibits myofibroblastic activation of valvular interstitial cells.

Heart valve disease is a growing problem worldwide. Though very common in older adults, the mechanisms behind the development of the disease aren't well understood, and at present the only therapeutic option is valve replacement. Valvular interstitial cells (VICs) may hold the answer. These cells can undergo pathological differentiation into contractile myofibroblasts or osteoblasts, leading to thickening and calcification of the valve tissue. Our study aimed to characterise the effect of fibroblast growth factor 2 (FGF-2) on the differentiation potential of VICs. We isolated VICs from diseased human valves and treated these cells with FGF-2 and TGF-β to elucidate effect of these growth factors on several myofibroblastic outcomes, in particular immunocytochemistry and gene expression. We used TGF-β as a positive control for myofibroblastic differentiation. We found that FGF-2 promotes a 'quiescent-type' morphology and inhibits the formation of α-smooth muscle actin positive myofibroblasts. FGF-2 reduced the calcification potential of VICs, with a marked reduction in the number of calcific nodules. FGF-2 interrupted the 'canonical' TGF-β signalling pathway, reducing the nuclear translocation of the SMAD2/3 complex. The panel of genes assayed revealed that FGF-2 promoted a quiescent-type pattern of gene expression, with significant downregulations in typical myofibroblast markers α smooth muscle actin, extracellular matrix proteins, and scleraxis. We did not see evidence of osteoblast differentiation: neither matrix-type calcification nor changes in osteoblast associated gene expression were observed. Our findings show that FGF-2 can reverse the myofibroblastic phenotype of VICs isolated from diseased valves and inhibit the calcification potential of these cells.

EPO synthesis induced by HIF-PHD inhibition is dependent on myofibroblast transdifferentiation and colocalizes with non-injured nephron segments in murine kidney fibrosis.

Erythropoietin (EPO) is regulated by hypoxia-inducible factor (HIF)-2. In the kidney, it is produced by cortico-medullary perivascular interstitial cells, which transdifferentiate into collagen-producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF-PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis. We investigated Epo transcription in myofibroblasts andcharacterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF-PHI molidustat. Lectin absorption chromatography was used to assess liver-derived EPO. In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy. In AN, molidustat-induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α-smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin-expressing, kidney injury molecule 1-negative nephron segments and contingent on residual renal function. Liver-derived EPO did not contribute to serum EPO in molidustat-treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy. Our studies suggest that HIF-PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.