BACKGROUND/AIM:Hereditary hemorrhagic telangiectasia (HHT) is a rare disease with an incidence of 1:5,000. HHT is inherited in an autosomal dominant manner and is associated with vascular malformations. It particularly affects the genes ENG (HHT1) and ACVRL1 (HHT2). Clinically, patients typically exhibit pronounced recurrent epistaxis. The aim of this study was to evaluate if ACVRL1 knockdown in HMEC-1 endothelial cells could induce a HHT2-like phenotype which could be deployed as a model for HHT2. MATERIALS AND METHODS:The human immortalized endothelial cell line HMEC-1 was used for the experiments. RNAi knockdown was performed using a pool of four siRNAs targeting the ACVRL1 gene. The gene knockdown was verified using RT-qPCR and Western blot analysis. The effects of ACVRL1 knockdown on angiogenesis were compared to a non-target (NT) small RNA control using a tube formation assay. The expression of 84 endothelia-associated genes was analyzed with RT-qPCR. RESULTS:Tube formation ability was significantly affected by ACVRL1 knockdown. In particular, the parameters total tube length, total segment length, total master segment length, total branching length, total mesh area and branching interval were significantly increased whereas total isolated branch length, number of master junctions, number of branches and the number of isolated segments decreased after ACVRL1 knockdown. Significant changes in the expression of angiogenesis related genes were detected by qPCR analysis and discussed. CONCLUSION:Knockdown of ACVRL1 in HMEC-1 endothelial cells leads to pathological angiogenesis with enhanced tube formation capacity, which is similar to the angiogenesis in vivo in patients with HHT2. The presented HMEC-1 cell-based system therefore has the potential to be deployed as an in vitro model for HHT2 studies.

Vascular malformations, including arteriovenous malformations (AVMs), venous malformations (VMs), and lymphatic malformations (LMs), carry a risk of rupture, bleeding, and significant morbidity. AVMs are high-flow lesions associated with abnormal shunting between arteries and veins, while VMs and LMs are low-flow malformations characterized by dilated venous or lymphatic channels. Current therapeutic options are limited, which often results in mental health issues and diminished quality of life, particularly in younger patients. Sclerotherapy with ethanol-based agents is an effective minimally invasive treatment, particularly for low-flow lesions. Injectable ethyl cellulose-based gels containing ethanol have emerged as a potential treatment for sclerosing these abnormal vessels; however, their exact mechanism of action and behavior within the vasculature remain inadequately understood. Comprehensive knowledge of the therapeutic mechanism is crucial for optimizing its clinical application and achieving the best treatment outcomes. In this study, we explored the production process, stability, and intravascular behavior of ethanol-based gels, providing essential information for clinical practice and patient care. We developed an injection model to elucidate the physical properties and dynamics of the gel under flow conditions in vitro and within blood vessels in ovo. Our results demonstrate an effective and safe injection, showing limited adverse effects. These insights contribute to advancing injectable polymer-based gel formulations, offering improved options for treating vascular malformations.

INTRODUCTION:Bevacizumab, a vascular endothelial growth factor inhibitor, is used off-label for treatment of severe anaemia related to epistaxis, gastrointestinal bleeding and/or severe hepatic arteriovenous malformations (HAVM) and right-sided cardiac failure in patients with hereditary haemorrhagic telangiectasia (HHT). AIM:To report the experience of treatment with bevacizumab within European Reference Network for Rare Multisystemic Vascular Diseases (VASCERN) centres. METHOD:A retrospective analysis of the usage, availability, efficacy and safety in HHT patients treated with bevacizumab in European VASCERN centres. RESULTS:A total of 151 patients received treatment with bevacizumab in European VASCERN centres. Most patients were treated in Denmark, France and the Netherlands. There was an improvement in haemoglobin with a mean increase of 2.9 g/dL (95% confidence interval 2.4, 3.6) and significant reduction in the number of red blood cell transfusions. Cardiac output was measured and improved in 85% of patients treated for high output cardiac failure in relation to severe HAVM. No severe adverse events were recorded, but 70 patients experienced at least one adverse event. CONCLUSION:Although this is a retrospective study, we demonstrated convincing efficacy of bevacizumab in the treatment of patients with HHT, with a good risk-benefit balance.

Einleitung Bei der Parotidektomie wird am häufigsten die bipolare Koagulation (BK) für die Hämostase verwendet. Dies ist für die Visualisierung des Gesichtsnervs und seiner Äste wichtig. Bisher liegen keine Daten zum sicheren Abstand vom Gesichtsnerv zur Durchführung der BK vor.

Introduction Head and neck squamous cell carcinomas (HNSCC) are the most prevalent malignancies of the upper aero-digestive tract. Resistance of HNSCC to radiotherapy hampers the efficacy of tumor treatment. The GSH/GSSG cellular redox buffer system is capable of neutralizing radicals such as radiation-induced reactive oxygen species thereby helping to protect tumor cells from therapy induced cell death.

Introduction Epistaxis is the most frequent symptom of hereditary hemorrhagic telangiectasia (HHT) and can severely affect quality of life. Until now, no optimal treatment method exists, therefore a multitude of different therapeutic approaches are applied. One option is the closure of the nasal nostrils, the so-called Young"s procedure. It has already been demonstrated that nasal blockage can worsen or induce obstructive sleep apnea (OSA) in healthy young women, however, no objective data existed if this would also apply to HHT patients which are more accustomed to nasal blockage by blood crusts and nasal packings.

Introduction HHT type 2 (HHT2) is an autosomal dominant inherited disease in which one allele of the ACVRL1 gene is mutant. Patients have severe disturbances in angiogenesis and clinically often present with severe nosebleeds and an impaired quality of life. The aim of our study was to investigate how overexpression of ACVRL1 in the endothelial cell line HMEC-1 affects angiogenesis.

Introduction Bipolar cautery (BC) is often performed in parotid surgery in order to achieve good hemostasis, which is important for the visualization of the facial nerve and its branches. To date, no data exists concerning an appropriate safe distance from facial nerve to perform BC.

The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild type SMAD4 cDNA was used to generate recombinant SMAD4 constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygous SMAD4 mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning of SMAD4 cDNA from the same cell line revealed an additional SMAD4 transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells.

Having suitable animal models is crucial to mimic human disease states and for the successful transfer of experimental data into clinical practice. In the field of papillomavirus research, the domestic rabbit (Oryctolagus cuniculus) has served as an indispensable model organism for almost 100 years. The identification and characterization of the first papillomaviruses in rabbits, their carcinogenic potential and their immunogenicity have contributed significantly to the state of knowledge on the genetics and life cycle of papillomaviruses in general, as well as the development of antiviral strategies such as vaccination procedures. Due to the high species specificity of papillomaviruses, only rabbit papillomaviruses (RPVs) can be used for animal studies on papilloma-based tumor diseases in the rabbit. The major focus of this article is on cottontail rabbit papillomavirus (CRPV)-related rabbit squamous cell carcinoma (RSCC). A brief history outlines the discovery and generation of experimentally used RSCC tumors. A comprehensive overview of the current CRPV-associated VX2 carcinoma-based tumor models with a major focus on human head and neck squamous cell carcinoma (HNSCC) tumor models is provided, and their strengths in terms of transferability to human HNSCC are discussed.

Background Tumor cells, able to provide sufficient ATP in response to irradiation, promote DNA damage repair and are therefore likely to survive the therapy. The aim of the present study was to better understand the metabolic energy response of HNSCC (head and neck squamous cell carcinoma) cells to irradiation, which could help to identify therapeutic weak points.

OBJECTIVE:Currently no data exist on what distance from facial nerve (FN) it is safe to perform bipolar cautery (BC) in parotid surgery, although frequently performed. METHODS:The degree of damage was measured using continuous intraoperative neuromonitoring (cIONM, NIM™ 3, Medtronic) in 16 Wistar rats. Amplitude drop of at least 50% (A50) or a loss of signal (LOS) in the cIONM was defined as harmful; BC was performed in power range 20-60 W. RESULTS:BC ≤30 W did not cause LOS (0/14 nerves). When applying 35 W, A50 occurred at 4 mm from FN and LOS was noted in 1 of 5 nerves. BC at a power of 40 to 60 W demonstrated LOS in all nerves (12/12) at a 5 mm distance. CONCLUSION:BC up to 30 W can be safely applied up to 3 mm distance from FN. 40 to 60 W should be avoided and used only at a distance of over 6 mm from FN. LEVEL OF EVIDENCE:NA/animal study. Laryngoscope, 135:1120-1126, 2025.

Next to alcohol and tobacco abuse, infection with human papillomaviruses (HPVs) is a major risk factor for developing head and neck squamous cell carcinomas (HNSCCs), leading to 350,000 casualties worldwide each year. Limited therapy options and drug resistance raise the urge for alternative methods such as photodynamic therapy (PDT), a minimally invasive procedure used to treat HNSCC and other cancers. We prepared lipid-coated polymeric nanoparticles encapsulating curcumin as the photosensitizer (CUR-LCNPs). The prepared CUR-LCNPs were in the nanometer range (153.37 ± 1.58 nm) and showed an encapsulation efficiency of 92.69 ± 0.03%. Proper lipid coating was visualized using atomic force microscopy (AFM). The CUR-LCNPs were tested in three HPVpos and three HPVneg HNSCC lines regarding their uptake capabilities and in vitro cell killing capacity, revealing a variable but highly significant tumor cell inhibiting effect in all tested HNSCC cell lines. No significant differences were detected between the HPVpos and HPVneg HNSCC groups (mean IC50: (9.34 ± 4.73 µmol/L vs. 6.88 ± 1.03 µmol/L), suggesting CUR-LCNPs/PDT to be a promising therapeutic option for HNSCC patients independent of their HPV status.

Supplementary Table 2A. Complete RT2 Profiler PCR Array 1. Overview of all genes analyzed by the RT2 Profiler PCR Array (SABiosciences, PANZ-052ZA). Fold changes of mRNA expression level measured in remitting VX2 tumors in the cause of the O3/O2-PP therapy (Regression, n=6) were calculated in reference to mRNA level measured in growing VX2 tumors of sham treated animals (Progression, n=6). Regulation of mRNA level is marked by ↑ (up) or ↓ (down). Statistic differences were calculated by the paired t-test and marked as: *p<0.05; **p<0.01; ***p<0.001; n.s. not significant.

Supplementary Table 1. Hemogram of rabbits, which respond to O3/O2-PP treatment by tumor clearance (n=7) and of rabbits, which showed a clear tumor progression after sham treatment (n=6). Blood parameters were measured before induction of the auricular VX2 tumor (base value) and of VX2 tumor bearing rabbits directly before and after the treatment schema. ‡ The base value includes blood parameter from animals of both treatment schema (n=13). Statistically significant changes between values before and after a treatment were marked with p<0.05. Abbreviations: WBC (White blood cells), RBC (Red blood cells), PLT (Platelets), HGB (Hemoglobin), HCT (Hematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration)

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

Background Hereditary hemorrhagic telangiectasia (HHT) type 2 occurs with a prevalence of 1:5,000; making it one of the more common rare diseases. HHT type 2 is an autosomal dominant inherited disease, with one allele of the ACVRL1 gene mutated. Patients present with severe disturbances of angiogenesis and very often exhibit clinically severe nosebleeds and impaired quality of life. The aim of our study was to observe how downregulation of ACVRL1 in the endothelial cell line HMEC-1 affects angiogenesis.

Background Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutant. Patients exhibit severe disturbances in TGF-beta/BMP dependent angiogenesis and, clinically, present with severe nosebleeds and a highly reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem (iPS) cells and evaluate the effects on TGF-beta and BMP related gene expression and angiogenesis.