Search Results (767)

We recently reported that phospholipase A2 (PLA2)-mediated production of prostaglandins within the ventromedial hypothalamus (VMH) plays a critical role in systemic glucose homeostasis. However, the role of PLA2 in the VMH in regulating food intake is still unclear. Here, we attempted to investigate the role of PLA2 in regulating food intake and body weight in male mice. We injected an adeno-associated virus encoding short hairpin RNA (AAV-shRNA) targeting cytosolic phospholipase A2 (shPla2g4a) into the VMH. We assessed food intake, body weight, oxygen consumption, glucose tolerance, and insulin sensitivity. Three weeks after the AAV injection, the shPla2g4a group exhibited increased food intake and body weight gain compared to controls (shSCRM). Energy expenditure, oxygen consumption, and respiratory quotient (RQ) were comparable between groups. Our findings suggest that the cPLA2-mediated pathway in the VMH is critical for feeding behavior and maintaining energy homeostasis. Further investigation is needed to elucidate the underlying mechanisms. Full article

Gene replacement using adeno-associated viral (AAV) vectors has become a major therapeutic avenue for neurodegenerative diseases (NDD). In single-gene diseases with loss-of-function mutations, the objective of gene therapy is to express therapeutic transgenes abundantly in cell populations that are implicated in the pathological phenotype. X-ALD is one of these orphan diseases. It is caused by ABCD1 gene mutations and its main clinical form is adreno-myelo-neuropathy (AMN), a disabling spinal cord axonopathy starting in middle-aged adults. Unfortunately, the main cell types involved are yet poorly identified, complicating the choice of cells to be targeted by AAV vectors. Pioneering gene therapy studies were performed in the Abcd1^-/y mouse model of AMN with AAV9 capsids carrying the ABCD1 gene. These studies tested ubiquitous or cell-specific promoters, various routes of vector injection, and different ages at intervention to either prevent or reverse the disease. The expression of one of these vectors was studied in the spinal cord of a healthy primate. In summary, gene therapy has made promising progress in the Abcd1^-/y mouse model, inaugurating gene replacement strategies in AMN patients. Because X-ALD is screened neonatally in a growing number of countries, gene therapy might be applied in the future to patients before they become overtly symptomatic. Full article

Cardiovascular disease encompasses a wide group of conditions that affect the heart and blood vessels. Of these diseases, cardiomyopathies and arrhythmias specifically have been well-studied in their relationship to cardiac dyads, nanoscopic structures that connect electrical signals to muscle contraction. The proper development and positioning of dyads is essential in excitation–contraction (EC) coupling and, thus, beating of the heart. Three proteins, namely CMYA5, JPH2, and BIN1, are responsible for maintaining the dyadic cleft between the T-tubule and junctional sarcoplasmic reticulum (jSR). Various other dyadic proteins play integral roles in the primary function of the dyad—translating a propagating action potential (AP) into a myocardial contraction. Ca²⁺, a secondary messenger in this process, acts as an allosteric activator of the sarcomere, and its cytoplasmic concentration is regulated by the dyad. Loss-of-function mutations have been shown to result in cardiomyopathies and arrhythmias. Adeno-associated virus (AAV) gene therapy with dyad components can rescue dyadic dysfunction, which results in cardiomyopathies and arrhythmias. Overall, the dyad and its components serve as essential mediators of calcium homeostasis and excitation–contraction coupling in the mammalian heart and, when dysfunctional, result in significant cardiac dysfunction, arrhythmias, morbidity, and mortality. Full article

Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) is a heterogeneous group of small-vessel vasculitides, characterized by the presence of antibodies binding to myeloperoxidase (MPO) and proteinase-3 (PR3) found in neutrophil granules. Apart from being the target of ANCA, neutrophils actively contribute to the vicious cycle of inflammation and vascular damage in AAV. On the other hand, platelets have recently been recognized as essential for thrombosis and as inflammatory effectors that collaborate with neutrophils, reinforcing the generation of reactive oxygen species (ROS) and the formation of neutrophil extracellular traps (NETs) in those diseases. Neutrophils exhibit morphological and functional heterogeneity in AAV, reflecting the complexity of their contribution to disease pathogenesis. Since long-term immunosuppression may be related to serious infections and malignancies, there is an urgent need for reliable biomarkers of disease activity to optimize the management of AAV. This review summarizes the current understanding of the role of neutrophils and platelets in the pathogenesis of granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), focusing on their crosstalk, and highlights the potential for identifying novel biomarkers relevant for predicting the disease course and its relapses. Full article

Background and objective. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) affects small- to medium-sized vessels and is characterized by the production of ANCAs. The ANCA-negative term is used if the patient otherwise fulfills the definition for AAV but has negative results on serologic testing for ANCAs. The objective of this study was to compare ANCA-positive and -negative vasculitis patients and to evaluate the main differences possibly related to the presence of ANCAs. Material and methods. A cross-sectional study of 73 patients treated at the tertiary Rheumatology Centre of University Hospital from the 1 January, 2001, to the 31August, 2023, with diagnoses of AAV was carried out. Clinical characteristics and laboratory data were collected at the onset or at the first year of the disease. Results. Forty-eight (65.8%) patients were ANCA-positive, while twenty-five (34.3%) were ANCA-negative. Distribution by gender was similar in both groups, with a female–male ratio of 2:1. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were elevated for all AAV patients, but values were higher in the ANCA-positive patients’ group. The median hemoglobin was 106 g/L in the seropositive group and 127 g/L in the seronegative group. A higher prevalence of kidney involvement (60.4%) with elevated serum creatinine level (93.5 µmol/L) was observed in the ANCA-positive group compared with 24% and 70 µmol/l in the ANCA-negative group (p < 0.05). Neurological involvement was more frequently found in the ANCA-positive patient group, too: 29.2% compared to 20%. Among patients with ANCA-negative vasculitis, 88% had pulmonary; 92% ear, nose, throat (ENT); 48% joint; and 28% skin presentation. In comparison, involvement of these organs was less common in the ANCA-positive patients’ group, at 79.2%, 60.4%, 31.3%, and 25 %, respectively. Conclusions. ANCA-positive patients appear to be in a more difficult clinical situation in terms of organ involvement and laboratory changes. Full article

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), including granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA), represent a spectrum of systemic disorders characterized by necrotizing inflammation of small- to medium-sized vessels. Nailfold videocapillaroscopy (NVC) is a validated, non-invasive technique routinely employed in the assessment of microvascular involvement in systemic sclerosis and in the differential diagnosis of Raynaud’s phenomenon; its application in the context of AAV, particularly EGPA, has not been investigated yet. The present study aims to assess the presence and the possible pattern of microcirculatory abnormalities detected by NVC in EGPA patients, and to explore potential correlations between capillaroscopic findings and disease activity status. Methods: A total of 29 patients with EGPA (19 women and 10 men), aged between 51 and 73 years, and 29 age- and sex-matched healthy controls were retrospectively enrolled between October 2023 and April 2025, after providing informed consent and meeting the inclusion and exclusion criteria. NVC was conducted in both groups to assess various morphological parameters, and mean capillary density was also calculated. Results: This study observed the presence of capillaroscopic alterations in the EGPA group, including decreased capillary density (38%), neoangiogenesis (72%), rolling (100%), pericapillary stippling (66%), and inverted capillary apex (52%). Overall, when comparing healthy controls with EGPA patients, microcirculatory abnormalities were significantly more prevalent in the latter. Specifically, scores for neoangiogenesis, capillary rolling, pericapillary stippling, and inverted capillary apex showed p-values < 0.001. Conclusions: Our study demonstrates a higher prevalence of four nailfold videocapillaroscopic abnormalities in patients with EGPA compared to healthy controls. However, the identification of these capillaroscopic alterations as specific to EGPA requires further confirmation. Ongoing studies aim to explore the potential role of NVC as a diagnostic marker and to investigate its correlation with the clinical manifestations of EGPA. Full article

Atherosclerosis is a complex disease characterized by pathological thickening of the arterial intima. The mechanisms underlying the induction and progression of atherosclerosis are convoluted and remain under active investigation, with key components such as lipid accumulation and local inflammation being identified. Several risk factors (e.g., metabolic disorders, genetic background, diet, infections) have been shown to exacerbate disease progression, but their roles as clinically relevant markers remain to be established. Despite the growing body of evidence on the molecular pathogenesis of atherosclerosis, there is no effective preventive treatment against the development of this disease. In this review, we focus on gene targets for gene therapy as a novel potential approach to cure and prevent atherosclerosis. We critically review recent research demonstrating the therapeutic potential of viral vector-based (adeno-associated virus (AAV) and lentivirus) gene therapy for the treatment of atherosclerosis. We also summarize alternative gene targets and approaches (e.g., non-coding RNA (ncRNA), micro RNA (miRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO), CRISPR/Cas9) that aim to limit disease progression. We highlight the importance of local inflammation in the pathogenesis of atherosclerosis and propose gene targets with anti-inflammatory activity to inhibit the pathological inflammatory response. In addition, we provide perspectives on the future development of gene therapeutics and their potential applications. We anticipate that recent advances in gene therapy will help to identify new and effective targets to prevent atherosclerosis. Full article

Heart failure (HF) has become an epidemic, with a prevalence of ~7 million cases in the USA. Despite accounting for nearly 50% of all HF cases, heart failure with a preserved ejection fraction (HFpEF) remains challenging to treat. Common pathophysiological mechanisms in HFpEF include oxidative stress, microvascular dysfunction, and chronic unresolved inflammation. Our lab focuses on oxidative stress-mediated cellular dysfunction, particularly the toxic effects of lipid peroxidation products like 4-hydroxy-2-nonenal (4HNE). Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme, plays a vital role in detoxifying 4HNE and thereby protecting the heart against pathological stress. ALDH2 activity is reduced in various metabolic stress-mediated cardiac pathologies. The dysfunction of coronary vascular endothelial cells (CVECs) is critical in initiating HFpEF development. Thus, we hypothesized that ectopic overexpression of ALDH2 in CVECs could mitigate metabolic stress-induced HFpEF pathogenesis. In this study, we tested the efficacy of intracardiac injections of the ALDH2 gene into CVECs in db/db mice—a model of obesity-induced type 2 diabetes mellitus (T2DM)—and their controls, db/m mice, by injection with ALDH2 constructs (AAV9-VE-cadherin-hALDH2-HA tag-P2A) or control constructs (AAV9-VE-cadherin-HA tag-P2A-eGFP). We found that intracardiac ALDH2 gene transfer increased ALDH2 levels specifically in CVECs compared to other myocardial cells. Additionally, we observed increased ALDH2 levels and activity, along with decreased 4HNE adducts, in the hearts of mice receiving ALDH2 gene transfer compared to control GFP transfer. Furthermore, ALDH2 gene transfer to CVECs improved diastolic function compared to GFP control alone. In conclusion, ectopic ALDH2 expression in CVECs can contribute, at least partially, to the amelioration of HFpEF. Full article

Adeno-associated virus (AAV) vectors have emerged as powerful tools for in vivo gene therapy, enabling long-term transgene expression in targeted tissues with minimal pathogenicity. This review examines the AAV serotypes used in clinical gene therapy trials for neurodegenerative (central nervous system, CNS) diseases, highlighting their tropisms, engineering advances, and translational progress. We discuss how capsid modifications, cell-specific promoters, and novel delivery routes are enhancing AAV tropism and reducing immunogenicity to overcome current limitations. Key clinical trials in neurodegenerative disorders (such as Parkinson’s, Alzheimer’s, and Huntington’s disease) are summarized, including delivery methods (intravenous, intracoronary, intrathecal, etc.) and outcomes. We further outline the regulatory landscape with recent approvals of AAV-based therapies and ongoing efforts to address safety challenges like immune responses and vector dose toxicity. A more translational, forward-looking perspective is adopted to consider combination therapies (e.g., AAV with immune modulation or genome editing) and strategic directions to improve the next generation of AAV vectors. Overall, continued innovation in AAV vector design and delivery, alongside careful clinical evaluation, is accelerating the translation of gene therapies for neurodegenerative diseases. Full article

Adeno-associated virus (AAV) vectors face a critical translational challenge in ocular gene therapy due to pre-existing neutralizing antibodies (NAbs) whose seroprevalence limits patient eligibility. Standard NAb detection using non-ocular cell models (Human Embryonic Kidney 293T) may inadequately predict retinal transduction inhibition due to cell type-related variations in receptor usage and immunogenicity. This study established parallel NAb detection platforms utilizing human retinal pigment epithelial (ARPE-19) cells and standard 293T cells to systematically evaluate clinical serum samples against ophthalmologically relevant AAV serotypes (2, 5, 8, 9) via luciferase reporter-based transduction inhibition assays. Comparative analysis demonstrated ARPE-19 exhibited 42–48% higher NAb titers against AAV5/9 compared to 293T cells, with distinct serotype-biased neutralization hierarchies observed between cellular models. Furthermore, female-derived sera exhibited significantly elevated NAbs against particular serotypes in the ARPE-19 system. Critically, inter-serotype cross-neutralization correlation patterns differed substantially between cellular platforms. These findings demonstrate that physiologically relevant retinal cellular models provide essential immunological profiling data, revealing NAb characteristics obscured in standard assays. Consequently, employing retinal cell-based platforms is crucial for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes in ocular gene therapy. Full article

Background/Objectives: Contextual memory associated with methamphetamine (METH) use contributes to relapse and persistence of addiction. Angiotensin II type 1 receptor (AT1R) signaling has been implicated in drug reinforcement. LCZ696, a clinically used combination of sacubitril (a neprilysin inhibitor) and valsartan (an AT1R antagonist), may interfere with METH-associated memory through the modulation of dopaminergic pathways. Methods: Male C57BL/6J mice were tested in a conditioned place preference (CPP) paradigm to assess the effects of LCZ696, sacubitril (AHU377), and valsartan on METH-induced memory expression and reinstatement. Synaptic plasticity in the nucleus accumbens (NAc) was examined by assessing the levels of synaptophysin (Syp) and postsynaptic density protein 95 (Psd95), as well as dendritic spine density. Dopaminergic signaling in the ventral tegmental area (VTA) was evaluated via ELISA, Western blotting, and chromatin immunoprecipitation (ChIP), targeting cAMP response element-binding protein (Creb) binding to the tyrosine hydroxylase (Th) promoter. To further assess the role of Th, an adeno-associated virus (AAV9) carrying a CRISPR-Cas9-based sgRNA targeting Th (AAV9-Th-sgRNA) was microinjected into the VTA. Results: LCZ696 and valsartan significantly reduced METH-induced CPP and reinstatement. LCZ696 reversed METH-induced synaptic and dopaminergic alterations and suppressed Creb-mediated Th transcription. Th knockdown attenuated both CPP acquisition and relapse. Conclusions: LCZ696 disrupts METH-associated contextual memory by modulating dopaminergic signaling and Creb-dependent Th expression, supporting its potential as a treatment for METH use disorder. Full article

Background/Objectives: Long non-coding RNAs (lncRNAs) play significant roles in tissue development and disease progression and have emerged as crucial regulators of gene expression. The hepatocyte nuclear factor alpha antisense RNA 1 (HNF1A-AS1) lncRNA is a particularly intriguing regulatory molecule in liver biology that is involved in the regulation of cytochrome P450 enzymes via epigenetic mechanisms. Despite the growing recognition of lncRNAs in liver disease, the comprehensive role of HNF1A-AS1 in liver function remains unclear. This study aimed to investigate the roles of the mouse homolog of the human HNF1A-AS1 lncRNA HNF1A opposite strand 1 (Hnf1aos1) in liver function, gene expression, and cellular processes using a mouse model to identify potential therapeutic targets for liver disorders. Methods: The knockdown of Hnf1aos1 was performed in in vitro mouse liver cell lines using siRNA and in vivo livers of AAV-shRNA complexes. Changes in the global expression landscapes of mRNA and proteins were revealed using RNA-seq and proteomics, respectively. Changes in the selected genes were further validated via real-time quantitative polymerase chain reaction (RT-qPCR). Phenotypic changes were assessed via histological and absorbance-based assays. Results: After the knockdown of Hnf1aos1, RNA-seq and proteomics analysis revealed the differential gene expression of the mRNAs and proteins involved in the processes of molecular transport, liver regeneration, and immune signaling pathways. The downregulation of ABCA1 and SREBF1 indicates their role in cholesterol transport and fatty acid and triglyceride synthesis. Additionally, significant reductions in hepatic triglyceride levels were observed in the Hnf1aos1-knockdown group, underscoring the impact on lipid regulation. Notably, the knockdown of Hnf1aos1 also led to an almost complete depletion of CYP7A1, the rate-limiting enzyme in bile acid synthesis, highlighting its role in cholesterol homeostasis and hepatotoxicity. Histological assessments confirmed these molecular findings, with increased hepatic inflammation, hepatocyte swelling, and disrupted liver architecture observed in the Hnf1aos1-knockdown mice. Conclusions: This study illustrated that Hnf1aos1 is a critical regulator of liver health, influencing both lipid metabolism and immune pathways. It maintains hepatic lipid homeostasis, modulates lipid-induced inflammatory responses, and contributes to viral immunity, indirectly affecting glucose and lipid metabolic balance. Full article

Background/Objectives: The field of nephrology is increasingly embracing advanced treatments and clinical trials that focus on inhibiting specific components of the complement cascade, a key driver in complement-mediated kidney diseases. Materials and Methods: This review aims to summarize innovative therapies targeting various pathways, including the inhibition of the terminal part of the complement pathway (mainly C5), the alternative pathway (factor B inhibitors), and the lectin pathway (MASP inhibitors. C5 inhibitors play a critical role in preventing the formation of the membrane attack complex (MAC), offering effective solutions for conditions like atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH). Meanwhile, avacopan, a C5a receptor antagonist, addresses ANCA-associated vasculitis (AAV) by mitigating inflammation and enabling reduced reliance on corticosteroids. Similarly, narsoplimab, which inhibits MASP-2, targets the lectin pathway implicated in conditions such as aHUS. Iptacopan, a factor B inhibitor, focuses on the alternative pathway and demonstrates efficacy in managing C3 glomerulopathy (C3G). Results: A systematic review of complement-targeted therapies was conducted, analysing studies from 2013 to 2023 that address unmet medical needs in primary and secondary glomerular diseases. Conclusions: Our systematic review of complement-targeted therapies shows that these tailored and innovative treatments may specifically address unmet medical needs in primary and secondary glomerular diseases. Full article

Recombinant adeno-associated virus (rAAV) is the leading vector for gene replacement therapy; however, the roles and regulation of host proteins in rAAV production remain incompletely understood. In this comparative proteomic analysis, we focused on proteins in the nucleus, the epicenter of DNA uptake, transcription, capsid assembly, and packaging. HEK-293 cells were analyzed under the following three conditions: (i) untransfected, (ii) mock-transfected with the ITR and an unrelated plasmid, and (iii) triple-transfected with rAAV2 production plasmids. Cells were harvested at 24 and 72 h post-transfection, and nuclear fractions were processed using filter-aided sample preparation (FASP) followed by nano-scale liquid chromatography–tandem mass spectrometry (nLC-Orbitrap MS/MS). Across all samples, we identified 3384 proteins, revealing significant regulatory changes associated with transfection and rAAV production. Transfection alone accounted for some of the most substantial proteomic shifts, while rAAV production induced diverse regulatory changes linked to cell cycle control, structure, and metabolism. STRING analysis of significantly regulated proteins also identified an enrichment of those associated with the Gene Ontology (GO) term ‘response to virus’. Additionally, we examined proteins with reported relation to adenoviral components. Our findings help to unravel the complexity of rAAV production, identify interesting targets for further investigation, and may contribute to improving rAAV yield. Full article