Fibrotic Conditions Research Articles

Role of Inflammasomes in Keloids and Hypertrophic Scars-Lessons Learned from Chronic Diabetic Wounds and Skin Fibrosis.

Keloids and hypertrophic scars are pathological cutaneous scars. They arise from excessive wound healing, which induces chronic dermal inflammation and results in overwhelming fibroblast production of extracellular matrix. Their etiology is unclear. Inflammasomes are multiprotein complexes that are important in proinflammatory innate-immune system responses. We asked whether inflammasomes participate in pathological scarring by examining the literature on scarring, diabetic wounds (also characterized by chronic inflammation), and systemic sclerosis (also marked by fibrosis). Pathological scars are predominantly populated by anti-inflammatory M2 macrophages and recent literature hints that this could be driven by non-canonical inflammasome signaling. Diabetic-wound healing associates with inflammasome activation in immune (macrophages) and non-immune (keratinocytes) cells. Fibrotic conditions associate with inflammasome activation and inflammasome-induced transition of epithelial cells/endothelial cells/macrophages into myofibroblasts that deposit excessive extracellular matrix. Studies suggest that mechanical stimuli activate inflammasomes via the cytoskeleton and that mechanotransduction-inflammasome crosstalk is involved in fibrosis. Further research should examine (i) the roles that various inflammasome types in macrophages, (myo)fibroblasts, and other cell types play in keloid development and (ii) how mechanical stimuli interact with inflammasomes and thereby drive scar growth. Such research is likely to significantly advance our understanding of pathological scarring and aid the development of new therapeutic strategies.

The Role of Macrophages in Liver Fibrosis: New Therapeutic Opportunities.

Chronic inflammation is the hallmark of fibrotic disorders and is characterized by the activation of immune cells in the damaged tissues. Macrophages have emerged as central players in the fibrotic process since they initiate, sustain and amplify the inflammatory reaction. As regards the liver, distinct populations of phagocytic cells, like Kupffer cells and monocyte-derived macrophages, are indisputably key cells implicated in the pathogenesis of several chronic liver diseases. In this review, we summarize the current knowledge on the origin, role and functions of macrophages in fibrotic conditions, with a specific focus on liver fibrosis; then, we discuss some innovative therapeutic strategies targeting macrophages in fibrotic liver diseases.

Contribution of HLA and KIR Alleles to Systemic Sclerosis Susceptibility and Immunological and Clinical Disease Subtypes.

Systemic sclerosis (SSc) is an autoinflammatory, fibrotic condition of unknown aetiology. The presence of detectable autoantibodies against diverse nuclear antigens, as well as strong HLA associations with disease, suggest autoimmune involvement, however the links between endogenous and exogenous risk factors and SSc pathology remain undetermined. We have conducted a genetic analysis of HLA inheritance in two independent and meta-analysed cohorts of 1,465 SSc cases and 13,273 controls, including stratified association analyses in clinical and autoantibody positive subgroups of disease. Additionally, we have used patient genotypes to impute gene dosages across the KIR locus, encoding paired activating and inhibitory lymphocyte receptors for Class I HLA ligands, to conduct the largest analysis of KIR-HLA epistatic interactions in SSc to date. We confirm previous Class II HLA associations with SSc risk and report a new Class I association with haplotype HLA-B*44:03-HLA-C*16:01 at genome-wide significance (GWS). We further report statistically significant HLA associations with clinical and serological subtypes of disease through direct case-case comparison, and report a new association of HLA-DRB1*15:01, previously shown to bind topoisomerase-1 derived peptides, with anti-topoisomerase (ATA) positive disease. Finally, we identify genetic epistasis between KIRs and HLA class I ligands, suggesting genetic modulation of lymphocyte activation may further contribute to an individual’s underlying disease risk. Taken together, these findings support future functional investigation into endogenous immunological and environmental stimuli for disrupted immune tolerance in SSc.

Early-stage Dupuytren's disease treatment; a promising next step?

Dupuytren's disease is a common progressive fibrotic condition in which nodules in the palm of the hand and fingers can develop into fibrous cords. Over time, the fibrous cords can develop into flexion contractures, for which patients often seek treatment. The consensus is that Dupuytren's disease does not need treatment until contractures develop to greater than 30° at the metacarpophalangeal and 15° at the proximal interphalangeal joint.1 Treatment commonly consists of limited fasciectomy to remove the cord, needle aponeurotomy, or collagenase injection to weaken and break the cord.

Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells.

Recovery of the contractile function of the heart and the regeneration of the myocardium after ischemic injury are contemporary issues in regenerative medicine and cell biology. This study aimed to analyze early transcriptional events in cardiac tissue after infarction and to explore the cell population that can be isolated from myocardial tissue. We induced myocardial infarction in Wistar rats by permanent ligation of the left coronary artery and showed a change in the expression pattern of Notch-associated genes and Bmp2/Runx2 in post-MI tissues using RNA sequencing and RT-PCR. We obtained primary cardiac mesenchymal cell (CMC) cultures from postinfarction myocardium by enzymatic dissociation of tissues, which retained part of the activation stimulus and had a pronounced proliferative potential, assessed using a “xCELLigence” real-time system. Hypoxia in vitro also causes healthy CMCs to overexpress Notch-associated genes and Bmp2/Runx2. Exogenous activation of the Notch signaling pathway by lentiviral transduction of healthy CMCs resulted in a dose-dependent activation of the Runx2 transcription factor but did not affect the activity of the Bmp2 factor. Thus, the results of this study showed that acute hypoxic stress could cause short-term activation of the embryonic signaling pathways Notch and Bmp in CMCs, and this interaction is closely related to the processes of early myocardial remodeling after a heart attack. The ability to correctly modulate and control the corresponding signals in the heart can help increase the regenerative capacity of the myocardium before the formation of fibrotic conditions.

Histology of tendon and enthesis - suitable techniques for specific research questions.

The musculoskeletal system consists of different components comprising a wide range of tissue types, with tendons being one part. Tendon degeneration or rupture have a high prevalence in all age groups, often with poor outcomes of surgical treatment such as chronic pain and high re-tear rates. Therefore, much effort has been directed to further develop diagnostic and therapeutic methods as well as reconstruction techniques, including using adequate placeholders or implants. Diagnostic approaches and advanced stages of preclinical studies will inevitably include histological examination of the pathologically affected tissue. The present study presents adequate tendon-related, histological techniques, including the embedding of soft- and hard-tissue samples in different media. Consideration is also given to samples containing residual implant materials or having been subjected to standard staining protocols and immunohistochemical procedures. The study further examines cells and tendon structure to detect degenerative, fibrotic or inflammatory conditions and possible foreign-body responses to implanted materials. Infraspinatus tendons from preclinical studies carried on rat and sheep samples, as well as human biceps tendon samples, have been used as example materials.

Pan-Lysyl Oxidase Inhibitor PXS-5505 Ameliorates Multiple-Organ Fibrosis by Inhibiting Collagen Crosslinks in Rodent Models of Systemic Sclerosis.

Systemic sclerosis (SSc) is characterised by progressive multiple organ fibrosis leading to morbidity and mortality. Lysyl oxidases play a vital role in the cross-linking of collagens and subsequent build-up of fibrosis in the extracellular matrix. As such, their inhibition provides a novel treatment paradigm for SSc. A novel small molecule pan-lysyl oxidase inhibitor, PXS-5505, currently in clinical development for myelofibrosis treatment was evaluated using in vivo rodent models resembling the fibrotic conditions in SSc. Both lysyl oxidase and lysyl oxidase-like 2 (LOXL2) expression were elevated in the skin and lung of SSc patients. The oral application of PXS-5505 inhibited lysyl oxidase activity in the skin and LOXL2 activity in the lung. PXS-5505 exhibited anti-fibrotic effects in the SSc skin mouse model, reducing dermal thickness and α-smooth muscle actin. Similarly, in the bleomycin-induced mouse lung model, PXS-5505 reduced pulmonary fibrosis toward normal levels, mediated by its ability to normalise collagen/elastin crosslink formation. PXS-5505 also reduced fibrotic extent in models of the ischaemia-reperfusion heart, the unilateral ureteral obstruction kidney, and the CCl4-induced fibrotic liver. PXS-5505 consistently demonstrates potent anti-fibrotic efficacy in multiple models of organ fibrosis relevant to the pathogenesis of SSc, suggesting that it may be efficacious as a novel approach for treating SSc.

Anti-tumour necrosis factor therapy for early-stage Dupuytren's disease (RIDD): a phase 2b, randomised, double-blind, placebo-controlled trial.

BackgroundDupuytren’s disease is a common fibrotic condition that causes the fingers to flex irreversibly into the palm. Treatments for late-stage disease all have limitations, and there is no approved treatment for early-stage disease. We identified tumour necrosis factor as a therapeutic target in Dupuytren’s disease, and in a dose ranging trial found 40 mg adalimumab in 0·4 mL to be most efficacious. Here we aimed to assess the effects of intranodular injection of adalimumab in early-stage disease.MethodsIn this phase 2b, randomised, double-blind, placebo-controlled trial adults with early-stage Dupuytren’s disease and an established clinically distinct nodule with a clear history of progression in the preceding 6 months were recruited from two clinical centres in the UK and were randomly assigned 1:1 to receive four injections of adalimumab or saline every 3 months. Participants and assessors were masked. The primary outcome was nodule hardness measured with a durometer at 12 months. Data were analysed by linear mixed effects regression models in the intention-to-treat population with multiple imputation for missing primary outcome data. The trial is registered at the ISRCTN registry, ISRCTN 27786905 and is complete.FindingsBetween Feb 17, 2017, and Jan 11, 2019, 284 participants were screened in the UK and 140 were enrolled. 47 (34%) participants were female and 93 (66%) were male. Mean age of participants was 59·7 years (SD 10·0). Primary outcome data were available from 113 participants. Nodule hardness was lower (−4·6 AU [95% CI −7·1 to −2·2], p=0·0002) in the adalimumab compared with the saline group at 12 months. There were no related serious adverse events; the most common adverse events were minor injection site reactions.InterpretationIntranodular injections of adalimumab in participants with early-stage Dupuytren’s disease resulted in softening and reduction in size of the nodules. Longer follow-up would be required to assess the effect of tumour necrosis factor inhibition on disease progression, extension deficit and hand function.

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.

Differences in Patient Characteristics and Surgical Outcomes in Inpatient vs Outpatient Surgery for Peyronie's Disease: Contemporary Data from the National Surgical Quality Improvement Program Database

ABSTRACT Introduction Peyronie's disease (PD) is a fibrotic condition of the tunical layer of corporal penis that can cause deformity, erectile dysfunction, and reduced quality of life in men. Surgical therapy remains an integral treatment of the condition in severe cases. However, there is a paucity of data describing trends in surgical practices. Objective To characterize the differences between inpatient (IP) and outpatient (OP) practice patterns for PD surgery. We utilized the National Surgical Quality Improvement Program (NSQIP) database to identify and compare factors and outcomes associated with each approach. Methods NSQIP database was queried for men undergoing PD surgery between 2005-2019. Patients were classified as either OP (<24 hours) or IP(>24 hours). Baseline demographics, operating time, and complications were compared between the two groups. Multivariate logistic regression evaluated factors associated with discharge times and 30-day complications. Results A total of 924 patients were identified and classified as IP in 157 and OP in 767 patients. Operative time was shorter in OP (89 minutes) compared to IP (126 minutes, p<0.001). A statistically significant difference was noted in comorbidities favoring IP surgery between the groups including chronic obstructive pulmonary disease, anemia, and pre-operative dyspnea. The 30-day complication rates between both groups differed notably for any complication (9.6% IP, 1.6% OP, p< 0.001), any readmission (3.8% IP, 0.7% OP, p 0.005) superficial incisional surgical site infection (5.1% IP, 0.7% IP, p< 0.001), and unplanned intubation (1.3% IP, 0 % OP, p 0.029). Conclusions Surgical treatment for PD is increasingly utilized as an OP procedure. Patients with shorter operative times and fewer comorbidities appear to be the best suited candidates OP procedures. Beginning in 2015, trends began to shift toward OP procedures. In the era of increasing hospital costs, a shift toward OP surgery for PD has been rising. Disclosure No

A vasculature niche orchestrates stromal cell phenotype through PDGF signaling: Importance in human fibrotic disease

SignificanceTissue fibrotic diseases, for example of the liver and lung, represent a huge unmet medical need. In this study, using single-cell RNA sequencing, cytometry by time of flight (CyTOF), tissue imaging, and functional assays, we identify a complex vascular niche in Dupuytren's disease (DD), a common localized fibrotic condition of the palm, where early-disease-stage tissue can be accessed readily. We uncover a population of myofibroblast precursors within the pericyte compartment and demonstrate that the endothelium instructs the differentiation of functionally distinct stromal cells, thereby orchestrating discrete microenvironments in the fibrotic milieu. Together, these findings provide a basis for the concept of targeting blood vessel signaling to control the progression of human fibrosis.

609 Available non-invasive skin probes distinguish between normal skin and hypertrophic scar but not laser-treated scar

IntroductionSkin fibrosis is the most under-studied fibrotic condition, and as such, there are limited treatment options for hypertrophic scar (HTS). HTS is difficult to study because scars improve over time, thus making the distinction between natural improvements and interventions difficult. In addition, clinical, histologic, cellular, and molecular outcome metrics are not agreed upon among providers/researchers. It was hypothesized that a set of non-invasive skin probes would be able to distinguish between HTS and normal skin (NS), but not between HTS before and after treatment with fractional ablative laser, despite histologic-level evidence showing improvement with treatment.MethodsWounds were created and HTS were allowed to form (n=8 scars). At Day 77, HTS and NS (n=4) were assessed with non-invasive skin probes measuring elasticity and trans-epidermal water loss (TEWL). HTS were then treated with CO2 fractional ablative laser (FLSR) at 70 mJ, 250Hz, and 1% density. The same data, sample collection, and FLSR treatment was carried out at weeks 1, 2, 3 and 4. Formalin-fixed biopsies were processed and stained with H&E, Herovici, Masson’s Trichrome, and Picrosirius red stains. Collagen type and architecture were qualitatively evaluated. Rete ridge ratio (RRR) was calculated from H&E stains.ResultsAll stains distinguished between HTS and NS with ease. Herovici stain showed differences in collagen architecture and collagen type after treatment with FLSR. Pre laser treatment, elasticity was different between HTS and NS (290.50±32.90 N/m vs. 90.92±5.61 N/m, p< 0.001). However, elasticity did not change after FLSR at 1, 2, 3, or 4 weeks (248.17±48.54, 215.54±44.68, 295.29±37.5, 290.79±30.17, p >0.05). RRR was different between HTS and NS (1.22±0.11 vs. 1.51±0.03, p=0.08). FLSR induced a significant increase in RRR in HTSs (pre=1.3±0.1 vs. week 4=1.9±0.1, p< 0.05). Pre-treatment, TEWL was different between HTS and NS (12.96±1.99 g/m2h vs. 8.72±1.11 g/m2h, p=0.09). However, TEWL did not change after FLSR at 1, 2, 3, or 4 weeks (15.95±1.99, 20.35± 3.15, 15.9±1.96, 14.01±2.22, p >0.05).ConclusionsOutcome metrics that non-invasively measure the qualities of scar are critical for HTS research to evaluate treatment effectiveness. Currently available technologies distinguish between NS and HTS well. However, they are inadequate to distinguish scars pre- and post-treatment despite evidence at the histologic level that changes occur. New technologies should be developed that are able to more effectively demonstrate changes to HTS after treatment.

Generation of Complex Syngeneic Liver Organoids from Induced Pluripotent Stem Cells to Model Human Liver Pathophysiology.

The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.

401 - Feasibility of Constructive Shear Wave Interference Ultrasound Technology in Evaluating Skin Stiffness in Fibrotic Skin Conditions

401 - Feasibility of Constructive Shear Wave Interference Ultrasound Technology in Evaluating Skin Stiffness in Fibrotic Skin Conditions

Conditional deletion of myeloid-specific mitofusin 2 but not mitofusin 1 promotes kidney fibrosis

Macrophages exert critical functions during kidney injury, inflammation, and tissue repair or fibrosis. Mitochondrial structural and functional aberrations due to an imbalance in mitochondrial fusion/fission processes are implicated in the pathogenesis of chronic kidney disease. Therefore, we investigated macrophage-specific functions of mitochondrial fusion proteins, mitofusin (MFN)1 and MFN2, in modulating macrophage mitochondrial dynamics, biogenesis, oxidative stress, polarization, and fibrotic response. MFN1 and MFN2 were found to be suppressed in mice after adenine diet-induced chronic kidney disease, in transforming growth factor-beta 1-treated bone marrow-derived macrophages, and in THP-1-derived human macrophages (a human leukemic cell line). However, abrogating Mfn2 but not Mfn1 in myeloid-lineage cells resulted in greater macrophage recruitment into the kidney during fibrosis and the macrophage-derived fibrotic response associated with collagen deposition culminating in worsening kidney function. Myeloid-specific Mfn1 /Mfn2 double knockout mice also showed increased adenine-induced fibrosis. Mfn2-deficient bone marrow-derived macrophages displayed enhanced polarization towards the profibrotic/M2 phenotype and impaired mitochondrial biogenesis. Macrophages in the kidney of Mfn2-deficient and double knockout but not Mfn1-deficient mice exhibited greater mitochondrial mass, size, oxidative stress and lower mitophagy under fibrotic conditions than the macrophages in the kidney of wild-type mice. Thus, downregulation of MFN2 but not MFN1 lead to macrophage polarization towards a profibrotic phenotype to promote kidney fibrosis through a mechanism involving suppression of macrophage mitophagy and dysfunctional mitochondrial dynamics.

Pirfenidone as a potential antifibrotic injectable for Dupuytren’s disease

Dupuytren's disease is a progressive fibrotic condition of the hand that causes contracture of fingers in later stages. Our previous in vitro studies suggest that the transformation of fibroblasts to myofibroblasts induced by transforming growth factor-beta can be inhibited by the addition of the antifibrotic drug, pirfenidone (PFD). We hypothesize that the local delivery of PFD directly to nodules can potentially prevent the progression to cords and, furthermore, that injection of PFD after the resection of cords can limit the recurrence of the disease. The purpose of this research was to develop a PFD injectable solution and to assess its safety in mice. Based on preformulation observations, a sterile solution containing up to 8 mg/0.4 mL of PFD was prepared in a phosphate buffer with and without 15%v/v N-methyl-2-pyrrolidone. Accelerated stability studies suggested that the product should be kept at refrigerated temperature (2–8 °C) for long-term storage. Safety studies involving subcutaneous administration to mice showed that 2–4 mg of PFD in 0.4 mL aqueous buffer did not elicit a significant inflammatory reaction. However, 4 mg PFD in 0.4 mL (FB) of buffer: NMP cosolvent system led to a significant increase in the influx of inflammatory cells and 8 mg PFD (FA) in the cosolvent system was lethal to the animals.

Myositis Antibodies and Interstitial Lung Disease.

Interstitial lung disease (ILD) comprises a heterogeneous group of inflammatory and fibrotic conditions, often resulting in progressive lung function decline and increased mortality. Connective tissue disease (CTD) should be considered in all patients with ILD, as distinguishing between CTD-ILD and other forms of fibrotic lung disease has important therapeutic and prognostic implications. The idiopathic inflammatory myopathies (IIM) represent a CTD subtype of growing interest to ILD experts. The expansion and availability of myositis-specific and myositis-associated antibody testing has allowed for improved disease detection and characterization. In this review, we highlight the relationship between myositis antibodies and ILD. Select forms of IIM, such as the antisynthetase syndrome and clinically amyopathic dermatomyositis can present with rapidly progressive ILD, warranting timely disease diagnosis and management. Disease phenotypes, prevalence, laboratory testing, prognosis, and management strategies are described according to select myositis antibodies. Myositis antibodies provide valuable information for clinicians managing patients with ILD. This review aims to increase awareness of their role in disease detection, pathophysiology, and possibly therapeutics.

The ubiquitin‐proteasomal pathway in glaucomatous lamina cribrosa cells

AbstractPurposePrimary open‐angle glaucoma (POAG) is an age‐related fibrotic condition and a leading cause of blindness worldwide. POAG‐related damage is initiated within the lamina cribrosa (LC) region of the optic nerve head, driven by the pathological activation of resident LC cells. LC cells bear striking similarities to proliferative, apoptotic‐resistant myofibroblasts known to be responsible for organ fibrosis. Myofibroblast dysregulation is linked to targeted proteasomal degradation of p53 by the E3 ubiquitin‐protein ligase MDM2 (mouse‐double‐minute‐2) thus negating p53’s important regulatory role in cell‐cycle/apoptosis. This project aims to evaluate the role of p53, MDM2, and the ubiquitin‐proteasomal pathway in glaucomatous LC cells.MethodsPrimary human normal LC (NLC) and glaucoma LC (GLC) cells (n = 3 donors) were cultured under standard conditions and treated for 48 hours with RG‐7112 (p53‐MDM2 interaction inhibitor, Abcam). The p53‐MDM2 ubiquitin‐proteasomal pathway was analysed by real‐time polymerase chain reaction (qRT‐PCR) for gene expression and protein levels via western blotting.ResultsMDM2 gene expression levels were significantly elevated (p = 0.006) in GLC cells (1.00 ± 0.12) versus NLC cells (0.89 ± 0.08). p53‐MDM2 inhibitor RG‐7112 treatment caused a further significant (p = 0.001) increase in MDM2 transcription levels in GLC cells (1.17 ± 0.04). p53 transcription levels were equivocal between GLC cells (0.88 ± 0.09) and NLC cells (0.87 ± 0.08), with RG‐7112 treatment leading to a significant increase (p = 0.028) in p53 transcription levels in GLC cells (0.95 ± 0.06). Western blot analysis showed significant decreased protein expression levels of p53 (0.76 ± 0.09)(p = 0.047) and increased protein expression of MDM2 (1.57 ± 0.33)(p = 0.042) in GLC cells compared to NLC. Interestingly, p53‐MDM2 inhibitor RG‐7112 treatment increased p53 (1.14 ± 0.43)(p = 0.267) and decreased MDM2 protein expression levels (0.92 ± 0.25)(p = 0.06) in GLC cells.ConclusionsOur data suggest the ubiquitin‐proteasomal pathway is significantly dysregulated in GLC cells with MDM2 led p53 protein degradation negatively impacting its key role as “guardian of the genome”. Targeting the p53 ubiquitin‐proteasomal pathway in lamina cribrosa fibrosis may lead to future novel therapeutic interventions.

Destructive fibrotic teamwork: how both microenvironment stiffness and profibrotic interleukin 13 impair alveolar macrophage phenotype and function.

The pulmonary fibrotic microenvironment is characterized by increased stiffness of lung tissue and enhanced secretion of profibrotic soluble cues contributing to a feedback loop that leads to dysregulated wound healing and lung failure. Pinpointing the individual and tandem effects of profibrotic stimuli in impairing immune cell response remains difficult and is needed for improved therapeutic strategies. We utilized a statistical design of experiment (DOE) to investigate how microenvironment stiffness and interleukin 13 (IL13), a profibrotic soluble factor linked with disease severity, contribute to the impaired macrophage response commonly observed in pulmonary fibrosis. We used engineered bioinspired hydrogels of different stiffness, ranging from healthy to fibrotic lung tissue, and cultured murine alveolar macrophages (MH-S cells) with or without IL13 to quantify cell response and analyze independent and synergistic effects. We found that, while both stiffness and IL13 independently influence macrophage morphology, phenotype, phagocytosis and efferocytosis, these factors work synergistically to exacerbate impaired macrophage phenotype and efferocytosis. These unique findings provide insights into how macrophages in fibrotic conditions are not as effective in clearing debris, contributing to fibrosis initiation/progression, and more broadly inform how underlying drivers of fibrosis modulate immune cell response to facilitate therapeutic strategies.

Bioevaluation of magnetic mesoporous silica rods: cytotoxicity, cell uptake and biodistribution in zebrafish and rodents.

Mesoporous silica nanoparticles (MSN) characterized by large surface area, pore volume, tunable chemistry, and biocompatibility have been widely studied in nanomedicine as imaging and therapeutic carriers. Most of these studies focused on spherical particles. In contrast, mesoporous silica rods (MSR) that are more challenging to prepare have been less investigated in terms of toxicity, cellular uptake, or biodistribution. Interestingly, previous studies showed that silica rods penetrate fibrous tissues or mucus layers more efficiently than their spherical counterparts. Recently, we reported the synthesis of MSR with distinct aspect ratios and validated their use in multiple imaging modalities by loading the pores with maghemite nanocrystals and functionalizing the silica surface with green and red fluorophores. Herein, based on an initial hypothesis of high liver accumulation of the MSR and a future vision that they could be used for early diagnosis or therapy in fibrotic liver diseases; the cytotoxicity and cellular uptake of MSR were assessed in zebrafish liver (ZFL) cells and the in vivo safety and biodistribution was investigated via fluorescence molecular imaging (FMI) and magnetic resonance imaging (MRI) employing zebrafish larvae and rodents. The selection of these animal models was prompted by the well-established fatty diet protocols inducing fibrotic liver in zebrafish or rodents that serve to investigate highly prevalent liver conditions such as non-alcoholic fatty liver disease (NAFLD). Our study demonstrated that magnetic MSR do not cause cytotoxicity in ZFL cells regardless of the rods' length and surface charge (for concentrations up to 50 μg ml-1, 6 h) and that MSR are taken up by the ZFL cells in large amounts despite their length of ∼1 μm. In zebrafish larvae, it was observed that they could be safely exposed to high MSR concentrations (up to 1 mg ml-1 for 96 h) and that the rods pass through the liver without causing toxicity. The high accumulation of MSR in rodents' livers at short post-injection times (20% of the administered dose) was confirmed by both FMI and MRI, highlighting the utility of the MSR for liver imaging by both techniques. Our results could open new avenues for the use of rod-shaped silica particles in the diagnosis of pathological liver conditions.