Aniko Keller-Pinter

Disorders of the vocal folds, including atrophy, scarring, and paralysis, frequently result in glottic insufficiency, impaired phonation, and reduced quality of life. Injection laryngoplasty has become a cornerstone treatment for these conditions by restoring vocal fold volume and improving glottic closure through the injection of biomaterials into the thyroarytenoid muscle or paraglottic space. A variety of injectable biomaterials are currently available, including hyaluronic acid, calcium hydroxylapatite, human collagen, autologous fat or fascia, hematogenous biomaterials, and synthetic polymers such as polydimethylsiloxane. These materials differ considerably in their viscoelastic properties, biocompatibility, degradation kinetics, durability, and capacity for host tissue integration. Hyaluronic acid-based fillers closely mimic the mechanical properties of the vocal fold lamina propria and may support extracellular matrix remodeling, whereas autologous fat and fascia offer longer-lasting augmentation with variable long-term persistence. Despite significant clinical improvements in voice quality and glottis competence, their therapeutic effects remain largely structural, providing passive medialization without restoring native tissue microarchitecture or biomechanical function of the vocal folds. Consequently, next-generation biomaterials are increasingly being designed to combine mechanical support with regenerative functionality by promoting extracellular matrix remodeling, preserving mucosal wave propagation, and enhancing long-term tissue integration. This review provides a translational overview of injectable biomaterials for vocal fold augmentation, integrating their clinical performance with biomechanical properties, biological compatibility, and emerging regenerative strategies for functional vocal fold restoration.

Background: Laryngotracheal fibrosis is a rare but severe complication of prolonged intubation, leading to airway narrowing, respiratory distress, dysphonia, and, in advanced cases, life-threatening airway obstruction. Current treatments are primarily surgical, while pharmacologic approaches such as mitomycin C, corticosteroids, or 5-fluorouracil show inconsistent efficacy and potential toxicity. Thus, there remains a critical need for safe and effective antifibrotic therapies. Transforming growth factor-beta (TGF-β) is a key mediator of fibrosis, promoting fibroblast activation, migration, and expression of profibrotic markers such as alpha-smooth muscle actin (α-SMA). Objectives: This study aimed to evaluate the antifibrotic potential of tilorone dihydrochloride, a synthetic small molecule, in human respiratory fibroblasts in vitro. Methods: Fibrotic alterations were assessed in human laryngotracheal fibrosis tissue samples. An in vitro model using MRC-5 human lung-derived fibroblasts was employed to investigate the effects of tilorone. Molecular analyses (RT-qPCR, immunocytochemistry, western blotting) quantified mRNA and protein expression of key signaling markers. Cell proliferation and viability were performed to evaluate potential cytotoxic effects of tilorone. Functional assays, including wound scratch and single-cell tracking, assessed fibroblast motility, and atomic force microscopy (AFM) measured extracellular matrix elasticity. Results: Phosphorylation of Smad1/5/8, a key transcription factor in the bone morphogenetic protein (BMP) signaling, was reduced in both human laryngotracheal fibrosis tissue and TGF-β-treated MRC-5 fibroblasts. Tilorone treatment did not affect MRC-5 cell viability or proliferation but increased BMP2, BMP4, BMP7, and BMP14 (GDF5; Growth Differentiation Factor 5) mRNA expression, enhanced Smad1/5/8 phosphorylation, and suppressed TGF-β-induced Smad2/3 phosphorylation. Functionally, tilorone inhibited TGF-β-driven fibroblast migration and α-SMA expression, downregulated collagen I and III mRNA levels, and restored extracellular matrix elasticity, as confirmed by AFM. Conclusion: Tilorone counteracts TGF-β-mediated profibrotic signaling and restores BMP pathway activity in MRC-5 fibroblasts in vitro. These findings identify tilorone as a promising therapeutic candidate for fibrotic airway diseases such as laryngotracheal fibrosis.

Amyotrophic lateral sclerosis (ALS) is a multisystem progressive neurodegenerative disease. A recent theory of ALS onsetting pathogenesis proposed that the initiating primary damage is an acquired irreversible intrafusal proprioceptive terminal PIEZO2 channelopathy with underlying genetic and environmental risk factors. This Piezo2 channelopathy may also disrupt the ultrafast proton-based oscillatory signaling to motor neurons through vesicular transporter 1 (VGLUT1) and to the hippocampus through VGLUT2. As a result, it may gradually degenerate motor neurons in which process K_v1.2 ion channels are gradually depleted. It also gradually depletes heat shock transcription factor-1 (HSF-1) in the hippocampus, hence negatively affecting adult hippocampal neurogenesis. Syndecans, especially syndecan-3 (SDC3) in the nervous system, may act as critical players in the maintenance of the crosstalk between Piezo ion channels. Hence, our goal was to reanalyze the potential pathogenic gene variants from the cohort of our previous ALS study with a special focus on the aforementioned genes. Reanalysis of data formerly acquired by whole-exome sequencing of 21 non-related adult ALS patients was carried out with a focus on 28 genes. Accordingly, we identified charge-altering variants of SDC3 in 13 patients out of 21 that may contribute to the impairment of the Piezo crosstalk, and the progressive loss of the proposed proton-based signaling to motor neurons and to the hippocampus. A variant of uncertain significance was identified in the KCNA2 gene that may facilitate the faster loss of K_v1.2 ion function on motor neurons when Piezo2 channelopathy prevails. Not to mention that one variant was identified in the potassium current rectifying ion channels encoding KCNK1 and KCNK16 genes that may also propel the ALS disease process and provide the autoimmune-like pathogenic background. Moreover, Piezo2 channelopathy likely promotes diminishing HSF1 function in the hippocampus in the presence of the identified HSF1 variant. The current findings may support the ALS onsetting acquired irreversible Piezo2 channelopathy-induced pathogenesis. However, the preliminary nature of these findings needs validation and further functional studies on cohorts with a larger sample size in the future.

Skeletal muscle, constituting 40–50% of total body mass, is vital for mobility, posture, and systemic homeostasis. Muscle contraction heavily relies on ATP, primarily generated by mitochondrial oxidative phosphorylation. Mitochondria play a key role in decoding intracellular calcium signals. The endocannabinoid system (ECS), including CB₁ receptors (CB₁Rs), broadly influences physiological processes and, in muscles, regulates functions like energy metabolism, development, and repair. While plasma membrane CB₁Rs (pCB₁Rs) are well-established, a distinct mitochondrial CB₁R (mtCB₁R) population also exists in muscles, influencing mitochondrial oxidative activity and quality control. We investigated the role of mtCB₁Rs in skeletal muscle physiology using a novel systemic mitochondrial CB₁ deletion murine model. Our in vivo studies showed no changes in motor function, coordination, or grip strength in mtCB₁ knockout mice. However, in vitro force measurements revealed significantly reduced specific force in both fast-twitch (EDL) and slow-twitch (SOL) muscles following mtCB₁R ablation. Interestingly, knockout EDL muscles exhibited hypertrophy, suggesting a compensatory response to reduced force quality. Electron microscopy revealed significant mitochondrial morphological abnormalities, including enlargement and irregular shapes, correlating with these functional deficits. High-resolution respirometry further demonstrated impaired mitochondrial respiration, with reduced oxidative phosphorylation and electron transport system capacities in knockout mitochondria. Crucially, mitochondrial membrane potential dissipated faster in mtCB₁ knockout muscle fibers, whilst mitochondrial calcium levels were higher at rest. These findings collectively establish that mtCB₁Rs are critical for maintaining mitochondrial health and function, directly impacting muscle energy production and contractile performance. Our results provide new insights into ECS-mediated regulation of skeletal muscle function and open therapeutic opportunities for muscle disorders and aging.

The endocannabinoid system (ECS) is known to regulate crucial bodily functions, including healthy muscle activity. However, its precise roles in normal skeletal muscle function and the development of muscle disorders remain unclear. Previously, we developed a tamoxifen-inducible, skeletal muscle-specific CB₁ receptor knockdown (skmCB1-KD) mouse model using the Cre/LoxP system. In this study, we aimed to clarify the mechanisms behind the observed reduction in muscle force generation in these mice. To investigate this, we analyzed calcium dynamics following electrical stimulation-induced muscle fatigue, assessed store-operated calcium entry (SOCE), and performed functional analysis of mitochondrial respiration. Our findings suggest that the reduced muscle performance observed in vivo likely arises from interconnected alterations in ATP production by mitochondria. Moreover, in skmCB1-KD mice, we detected a significant decrease in a component of the respiratory chain (complex IV) and a slowed dissipation of mitochondrial membrane potential upon the addition of an un-coupler (FCCP).

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing and is caused by excessive fat deposition in the liver due to non-alcoholic factors. Aging is a major risk factor for the development and progression of MASLD. In this study, we investigated the metabolic effects of tilorone, a synthetic small molecule, in a high-fat diet (HFD) mouse model, with a focus on the liver function and signaling. We demonstrate that tilorone attenuated HFD-induced steatosis by restoring bone morphogenetic protein 9 (BMP9)-Smad1/5/8 signaling and upregulating peroxisome proliferator-activated receptor gamma (PPARγ) expression. Tilorone reduced HFD-induced increases in body weight, adipose tissue and liver weight, and blood glucose levels, and improved glucose tolerance in HFD mice. PET/MRI imaging demonstrated enhanced ¹⁸ FDG ( ¹⁸ F-fluoro-2-deoxyglucose) uptake in liver, skeletal muscle, adipose tissue, and myocardium of tilorone-treated HFD animals. Histological analysis showed that tilorone reduced the HFD-induced diffuse, macrovesicular steatosis (S3/3), and machine learning-based image analysis revealed a decrease in lipid droplet size and lipid content. HFD caused the disappearance of liver glycogen, but tilorone increased glycogen levels. High-resolution respirometry indicated that tilorone reduced HFD-induced increases in mitochondrial complex II-linked oxidative phosphorylation and complex IV activity. These findings revealed the beneficial effects of tilorone on HFD and highlight its therapeutic potential in MASLD, particularly given that tilorone is a synthetic small molecule and can be administered orally. Further studies are required to explore its clinical application.

The endocannabinoid system (ECS) regulates and controls many of our most critical bodily functions including healthy muscles however, its potential role(s) in physiological skeletal muscle function and its disorders are unclear. Earlier using the Cre/LoxP system we generated and characterized a tamoxifen-inducible skel-etal muscle-specific CB1R knockdown (skmCB1-KD) mouse model. Here, we sought to elucidate the mechanisms underlying reduced muscle force generation. To address this, we measured calcium movements following electrical stimula-tion-induced muscle fatigue, evaluated store-operated calcium entry (SOCE), and conducted functional analysis of respiring mitochondria. Based on our measure-ments, it is likely that the reduced muscle performance recorded in vivo may oc-cur due to complementary changes in ATP production by mitochondria. Further-more, in skmCB1-KD mice, we observed a significant decrease in one component of the respiratory chain and a delayed mitochondrial membrane potential dissipa-tion upon the application of an un-coupler.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, arises from skeletal muscle cells that fail to differentiate terminally. Two subgroups of RMS, fusion-positive and fusion-negative RMS (FPRMS and FNRMS, respectively), are characterized by the presence or absence of the PAX3/7-FOXO1 fusion gene. RMSs frequently exhibit increased expression of human epidermal growth factor receptor-2 (HER2). Trastuzumab is a humanized monoclonal antibody targeting HER2, and its potential role in RMS treatment remains to be elucidated. Syndecan-4 (SDC4) is a heparan sulfate proteoglycan (HSPG) affecting myogenesis via Rac1-mediated actin remodeling. Previously, we demonstrated that the SDC4 gene is amplified in 28% of human FNRMS samples, associated with high mRNA expression, suggesting a tumor driver role. In this study, after analyzing the copy numbers and mRNA expressions of other HSPGs in human RMS samples, we found that in addition to SDC4, syndecan-1, syndecan-2, and glypican-1 were also amplified and highly expressed in FNRMS. In RD (human FNRMS) cells, elevated SDC4 expression was accompanied by low levels of phospho-Ser179 of SDC4, leading to high Rac1-GTP activity. Notably, this high SDC4 expression in RD cells decreased following trastuzumab treatment. Trastuzumab decreased the levels of G1/S checkpoint regulators cyclin E and cyclin D1 and reduced the cell number; however, it also downregulated the cyclin-dependent kinase inhibitor p21. The level of MyoD, a transcription factor essential for RMS cell survival, also decreased following trastuzumab administration. Our findings contribute to the understanding of the role of SDC4 in FNRMS. Since HER2 is expressed in about half of RMSs, the trastuzumab-mediated changes observed here may have therapeutic implications.