Models Of Human Disease Research Articles

Topical ophthalmic instillation of engineered hMSCs-derived exosomes: A novel non-invasive therapeutic strategy for ocular posterior-segment disorder

Due to the presence of protective mechanisms and blood-ocular barriers in the eye, drugs aimed at treating posterior segment ophthalmic disorder have to be administrated mostly through periocular or intravitreal injection. In the current study, we sought to investigate whether topical ophthalmic instillation of human mesenchymal stem cells (hMSCs)-derived exosomes can prevent and treat experimental autoimmune uveitis (EAU), a posterior segment ophthalmic disease induced in animals and considered a model of human autoimmune diseases of the eye. Our studies reveal that topical ophthalmic instillation of hMSCs-derived exosomes can effectively ameliorate EAU. More importantly, we demonstrate that exosomes modified by trans-activator of transcription peptide (TAT) were more effective than naive exosomes in penetrating ocular barrier and preventing/treating EAU. Taken together, these results indicate that topical ophthalmic instillation of TAT-peptide modified exosomes represents a novel non-invasive therapeutic strategy for posterior-segment ophthalmic disorders.

Transcriptional regulation of hexokinase 2 by BRD4 drives perivascular adipose tissue meta-inflammation in cardiometabolic disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Holcim Stiftung SwissLife Stiftung Background/Introduction BRD4 is an epigenetic reader that modulates inflammatory transcriptional programmes implicated in cancer. The therapeutic potential of BRD4 modulation in cardiometabolic disorders remains elusive. Purpose We investigate BRD4-related transcriptional programmes and therapeutic modulation in mouse and human models of cardiometabolic disease. Methods Small arteries (100-300 μM) from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were dissected from visceral fat biopsies and mounted on a pressurised myograph to assess ex-vivo vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. Vasorelaxation was assessed in the presence or in the absence of perivascular adipose tissue (PVAT). An experimental mouse model of cardiometabolic disease (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) or vehicle for 10 days (n=6 each group) to test in vivo effect of chronic BRD4 inhibition on endothelial and PVAT functions. Vascular and PVAT levels of cytosolic and mitochondrial ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression were measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array and confirmed by ChIP, qPCR and Western blot and further characterised by metabolomics, lipidomics and mitochondrial swelling assays. Results Endothelial-dependent vasorelaxation and tissue levels of TNF-α, IL-1β and IL-6 were altered in the vessel wall and PVAT from cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction. Distinct ex-vivo modulation of well-established inflammatory pathways showed that the effect observed with BRD4 inhibition was stronger than with anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. (Figure 1). Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validated the findings, showing a PVAT glycolytic shift in condition of disease. Finally, ex-vivo selective inhibition of HK2 restored vascular dysfunction (Figure 2). Conclusions Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic-related vascular dysfunction reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammatory pathways might represent a promising strategy to prevent and vascular dysfunction, key signature of cardiometabolic disease.Figure 1Figure 2

Human in vitro disease modeling of ATTR cardiac amyloidosis

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Pfizer Alnylam Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive disease with deposition of TTR amyloid fibrils in the myocardium and cardiac dysfunction with dismal prognosis. In ATTR-CM, either genetic mutations (variant; v) or ageing (wild-type; wt) leads to TTR instability. Late diagnosis and a lack of human disease models limit disease understanding and therapeutic discovery. This study aimed to model ATTR-CM in vitro by exposing various cardiac cell types and multicellular 3D cardiac spheroids to TTR fibrils, characterizing disease phenotypes and providing insights into the cellular mechanisms of disease progression. Human recombinant TTR (WT, V30M, V122I) and acid induced fibrils were generated and characterized. Seeding hiPSC-cardiomyocytes (CMs) and endothelial cells (ECs) on TTR fibrils resulted in cell viability reduction. Confocal microscopy revealed the extracellular attachment of TTR fibrils, leading to sarcomere disorganization, prolonged calcium transients and disrupted electromechanical coupling in hiPSC-CMs, and declined migration capacity of ECs showing aberrant cell morphology. Similarly, cardiac spheroids incorporating TTR fibrils resulted in decreased cell viability and prolonged calcium transients with lower amplitude. Our models show the discovery of early cellular ATTR-CM phenotypes, providing a novel approach to study the disease progression, potentially facilitating the identification of novel therapeutic targets and biomarkers.Cytotoxicity of TTR fibrils on hiPS-CMs

VITAL A FISH: A CRITICAL REVIEW OF ZEBRAFISH MODELS IN DISEASE SCENARIO AND CASE REPORTS SCREENS

ABSTRACT Virtually every major medical advance of the last century and at still has depended upon research with animals. Zebrafish's journey from the ocean to the laboratory leads to major scientific breakthroughs. Transparency structure of zebrafish helps in monitoring their internal structures and are permitting scientist to see effectes of nano particles in fish. Their organs share the same main features as humans and so can be used to study human developmental processes. Zebrafish congruence 70% of their genes with humans, and 84% of ailment-depended genes have zebrafish congruence. The zebrafish embryos can also genetically modified. Certain fishes like zebrafish are able to regenerate damaged retinal nerve cells. Müller galia cells in retina of zebrafish can transform in response to injury and act like stem cells to regrow the retina and replace all damaged neurons. Though humans have the same exact Müller galia cell, they don’t respond to damaged in the same way. Zebrafish are also very responsive to having their genomes edited. Zebrafish regenerate some tissue such as heart in during larval stage. In additionaly zebrafish are used as an animal model to study pharmocology – how drugs work and what they do to an organism’s body. Aim of this review, here, we review current knowledge of how these specialized structures and model organism by focusing on cellular behaviors and molecular mechanisms, highlighting findings from in vivo models and briefly discussing the recent advances in tissue cell culture and organoids. Review discusses the applications of human organoids models of disease on model organism and outlines the ailment treatments.

Рroblems of diagnostics of dysfunctions of the olfactory analyzer of laboratory animals on the basis of behavioral and electrophysiological methods of research

Olfactory impairment (decreased acuity, impaired adequate identification of odorants) reduces the quality of life of patients and can be a symptom of a wide range of pathologies of the organism, in particular neurodegenerative processes in the brain. Quantitative measurement of olfactory acuity is necessary for diagnostics of olfactory dysfunctions, monitoring the dynamics of olfaction after pharmacological or surgical treatment. The searching for optimal methods of analyzing olfactory thresholds on animal models of human diseases accompanied by anosmia and comparing them with those in humans seems to be especially urgent problem at the moment. This is necessary for the selection of a valid animal model for the evaluation of new drugs and development the therapy for a wide range of pathologies. The review analyzes publications devoted to the study of diseases accompanied by anosmia or hyposmia, their zootropic models, and methods of olfactory function assessment. Models for COVID19, Alzheimer’s disease, Parkinson’s disease, diabetes types (1 and 2 type), Kalman syndrome, and Bardet-Biedl syndrome, for which olfactory dysfunction and/or defects of olfactory system are present, were analyzed. The review notes the paucity of data on the measurement of olfactory thresholds in model animals.

A Scalable Histological Method to Embed and Section Multiple Brains Simultaneously.

The preparation and processing of rodent brains for evaluation by immunohistochemistry is time-consuming. A large number of mouse brains are routinely used in experiments in neuroscience laboratories to evaluate several models of human diseases. Thus, methods are needed to reduce the time associated with processing brains for histology. A scalable method was developed to embed, section, and stain multiple mouse brains using supplies found in any common histology laboratory. Section collection schemes can be scaled to provide identical bregma locations between adjacent sections for immunohistochemistry, facilitating comprehensive, high-quality immunohistochemistry. As a result, sectioning and staining times are considerably reduced as sections from multiple blocks are stained simultaneously. This method improves on previous procedures and allows multiple embedding and subsequent immunostaining of brains easily with a dramatically reduced time requirement. Furthermore, we expand this method for use in numerous mouse tissues, rat brain tissue, and post-mortem human brain and arterial tissues. In summary, this procedure allows the processing of many rodent or human tissues from perfusion through microscopy in 10 days or less.

Leveraging dirty mice that have microbial exposure to improve preclinical models of human immune status and disease.

Leveraging dirty mice that have microbial exposure to improve preclinical models of human immune status and disease.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

Pathway-based, reaction-specific annotation of disease variants for elucidation of molecular phenotypes.

Germline and somatic mutations can give rise to proteins with altered activity, including both gain and loss-of-function. The effects of these variants can be captured in disease-specific reactions and pathways that highlight the resulting changes to normal biology. A disease reaction is defined as an aberrant reaction in which a variant protein participates. A disease pathway is defined as a pathway that contains a disease reaction. Annotation of disease variants as participants of disease reactions and disease pathways can provide a standardized overview of molecular phenotypes of pathogenic variants that is amenable to computational mining and mathematical modeling. Reactome (https://reactome.org/), an open source, manually curated, peer-reviewed database of human biological pathways, in addition to providing annotations for >11 000 unique human proteins in the context of ∼15 000 wild-type reactions within more than 2000 wild-type pathways, also provides annotations for >4000 disease variants of close to 400 genes as participants of ∼800 disease reactions in the context of ∼400 disease pathways. Functional annotation of disease variants proceeds from normal gene functions, described in wild-type reactions and pathways, through disease variants whose divergence from normal molecular behaviors has been experimentally verified, to extrapolation from molecular phenotypes of characterized variants to variants of unknown significance using criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Reactome's data model enables mapping of disease variant datasets to specific disease reactions within disease pathways, providing a platform to infer pathway output impacts of numerous human disease variants and model organism orthologs, complementing computational predictions of variant pathogenicity. Database URL: https://reactome.org/.

Severe cardiac and skeletal manifestations in DMD-edited microminipigs: an advanced surrogate for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an intractable X-linked muscular dystrophy caused by mutations in the DMD gene. While many animal models have been used to study the disease, translating findings to humans has been challenging. Microminipigs, with their pronounced physiological similarity to humans and notably compact size amongst pig models, could offer a more representative model for human diseases. Here, we accomplished precise DMD modification in microminipigs by co-injecting embryos with Cas9 protein and a single-guide RNA targeting exon 23 of DMD. The DMD-edited microminipigs exhibited pronounced clinical phenotypes, including perturbed locomotion and body-wide skeletal muscle weakness and atrophy, alongside augmented serum creatine kinase levels. Muscle weakness was observed as of one month of age, respiratory and cardiac dysfunctions emerged by the sixth month, and the maximum lifespan was 29.9 months. Histopathological evaluations confirmed dystrophin deficiency and pronounced dystrophic pathology in the skeletal and myocardial tissues, demonstrating that these animals are an unprecedented model for studying human DMD. The model stands as a distinct and crucial tool in biomedical research, offering deep understanding of disease progression and enhancing therapeutic assessments, with potential to influence forthcoming treatment approaches.

Gastric Organoid, a Promising Modeling for Gastric Stem Cell Homeostasis and Therapeutic Application.

The elucidation of the pathophysiology underlying various diseases necessitates the development of research platforms that faithfully mimic in vivo conditions. Traditional model systems such as two-dimensional cell cultures and animal models have proven inadequate in capturing the complexities of human disease modeling. However, recent strides in organoid culture systems have opened up new avenues for comprehending gastric stem cell homeostasis and associated diseases, notably gastric cancer. Given the significance of gastric cancer, a thorough understanding of its pathophysiology and molecular underpinnings is imperative. To this end, the utilization of patient-derived organoid libraries emerges as a remarkable platform, as it faithfully mirrors patient-specific characteristics, including mutation profiles and drug sensitivities. Furthermore, genetic manipulation of gastric organoids facilitates the exploration of molecular mechanisms underlying gastric cancer development. This review provides a comprehensive overview of recent advancements in various adult stem cell-derived gastric organoid models and their diverse applications.

Airway epithelial cell-specific deletion of EGFR in Scnn1b transgenic mice modulates inflammatory responses but doesn’t affect muco-obstructive features of lung disease

Abstract Epidermal growth factor receptor (EGFR) is known to regulate airway mucous cell metaplasia, but the roles of EGFR-mediated pathway in the manifestation of cystic fibrosis (CF)-relevant lung phenotypes remain unclear. We hypothesized that airway epithelial cell-specific deficiency of EGFR mitigates muco-obstructive and inflammatory responses in sodium channel, non-voltage gated 1, beta subunit (Scnn1b) overexpressing -transgenic (Tg+) mice, a mouse model of human CF-like lung disease. To test our hypothesis, we generated airway epithelial cell-specific EGFR-deficient mice and performed various endpoint analyses. The deletion of EGFR in airway epithelial cells in Tg+ mice resulted in increased neutrophilic and macrophage infiltration into lung airspaces, which was accompanied by significantly increased levels of KC, G-CSF, MIP-2, MIP-1α, and MIP-1β. Additionally, as compared to EGFR-sufficient Tg+ mice, airway epithelial cell-specific EGFR-deficient Tg+ mice exhibited significantly increased postnatal mortality, compromised bacterial clearance and elevated markers of Th2 inflammation, including Il13, Slc26a4, and Retnla. Counterintuitively, the deletion of EGFR in airway epithelial cells of Tg+ mice did not alter mucous cell metaplasia, MUC5AC/5B production, and degree of mucus obstruction. Our data shows that, unlike inflammation, muco-obstruction-relevant responses in Tg+ mice are not dependent on airway epithelial cell-specific EGFR signaling.

TRANSCRIPTIONAL REGULATION OF HEXOKINASE-2 BY BRD4 DRIVES PERIVASCULAR ADIPOSE TISSUE META-INFLAMMATION IN CARDIOMETABOLIC DISEASE

Objective: To investigate BRD4-related transcriptional programmes and therapeutic modulation in mouse and human models of cardiometabolic disease. Design and method: Small arteries (0.1-0.3 mm) from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were dissected from visceral fat biopsies and mounted on a pressurised myograph to assess the ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated, in the presence or in the absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. A cardiometabolic mouse (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) to test in vivo effect of chronic BRD4 inhibition. ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression were measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom qPCR array, confirmed by ChIP, qPCR and Western blot and further characterised by metabolomics, lipidomics and mitochondrial swelling assays. Results: Endothelial-dependent vasorelaxation and tissue levels of TNF-alpha, IL-1beta, and IL-6 were altered in the vessel wall and PVAT from cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater when compared to anti-IL-1beta, anti-IL-6 receptor and anti-TNF-alpha. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validate the findings, showing a PVAT glycolytic shift in condition of disease. Finally, ex-vivo selective inhibition of HK2 restored vascular dysfunction. Conclusions: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic-related vascular dysfunction reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammatory pathways might represent a promising strategy to prevent and reverse vascular dysfunction, key signature of cardiometabolic disease.

Is the Zucker Diabetic Fat Rat an Appropriate Model of Diabetic Kidney Disease: A Discussion on Hydronephrosis

Introduction: Kidney disease is a significant concern for global healthcare, particularly in individuals with diabetes. Experimental research into the pathology of diabetes is dependent on the utilisation of appropriate animal models. The Zucker Diabetic Fat (ZDF) rat has a Leptin receptor deficiency and is a commonly used model of type 2 diabetes. Here, we examine the relevance of this model for the investigation of diabetic kidney disease, with a particular focus on vascular imaging. The Zucker Lean (ZL) rat is a non-diabetic strain of the same rat lineage and will be used as a control. Method: This dataset used the ZDF rat (n=13) and the Zucker Lean (ZL) rat (n=16). Individuals were scanned at ages of 12 weeks (ZDF n=4, ZL n=4), 22 weeks (ZDF n=4, ZL n=5), and 40 weeks (ZDF n=5, ZL n=7). During open laparotomy, rats were maintained on a respirator under general anaesthesia via isoflurane and blood pressure was monitored via a central catheter. During surgery, a blood glucose measurement was taken from the tail vein and samples of arterial blood and urine were collected. The contrast medium Microfil (MV122, Flow Tech Inc., Carver, MA) was injected post-mortem via the renal artery and excised kidneys were scanned for 11 hours in a ZEISS XRadia 410 Versa μCT scanner (Carl Zeiss Microscopy GmbH, Jena, Germany). Results: As expected, the ZDF rats developed significant hyperglycaemia from 12 weeks, significant increases in albumin creatinine ratio (ACR) from 22 weeks, and a significant decrease in glomerular filtration rate (GFR) from 40 weeks. Concurrent hydronephrosis affecting more than 10% of total kidney volume was detected in 51% of all rats (group prevalence rates were ZDF=69% and ZL=38%). The percent of the kidney mass affected by hydronephrosis was significantly higher in ZDF than ZL (ANOVA row factor p=0,029*). Age had no significant impact on the size of the hydronephrotic cavity. There was a significant correlation between the degree of hyperglycaemia and the size of the hydronephrotic cavity (mm) (r=0.57, p=0,0014**). ACR, GFR and urine excretion rate were unaffected by hydronephrosis. Hydronephrosis did not have a significant impact on vascular density in the remaining kidney tissue (p=0.53). Conclusion: The high prevalence of hydronephrosis development in the ZDF rat questions its appropriateness to model human diabetic kidney disease, as hydronephrosis is not commonly seen in diabetic patients. While vascular density was preserved in the remaining kidney tissue, the huge loss of vascular tissue caused by hydronephrotic erosion means this model is limited for all renal imaging applications. Both ZDF and ZL rats develop hydronephrosis suggesting this may be a genetic condition of their common lineage. However, the conditions' increased severity in the ZDF rat indicates that the deficit in the leptin receptor contributes to disease pathology. The significant relationship between hyperglycaemia and hydronephrosis size suggests the two conditions are interacting, raising questions about whether the underlying molecular mechanisms of this model of diabetic kidney disease have any relevance for translational research. Funded by European Research Council Synergy grant SURE, project no. 854796. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Salt-Sensitive Hypertension and Renal Damage are Amplified by Adoptive Transfer of CD4+ and Not CD8+ T Cells: A Role of NADPH Oxidase 2 (NOX2)

Infiltration of T cells into the kidney of the Dahl Salt Sensitive (SS) rat model of human disease accompanies the development of salt-sensitive hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SSCD247−/−) attenuates hypertension and renal damage, and subsequent replacement of T cells by splenocyte transfer restores the disease phenotype. The present studies were performed to assess if the T cell subtype (CD4+ vs CD8+) is important in Dahl SS hypertension and interrogate the mechanism of action. In initial experiments, adoptive transfer of purified CD4+ or CD8+ T cells (~5 million) from splenocytes isolated from the Dahl SS (CD4+ SS→CD247 or CD8+ SS→CD247) or PBS vehicle (PBS→CD247) into the SSCD247−/− rat was performed on postnatal day 5. Animals were recovered and instrumented with radiotelemeters at 7 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→CD247, CD8+ SS→CD247, or PBS→CD247 groups (113±1, 115±1, 114±1 mmHg; n=21, 10, 24, respectively). After three weeks of HS, rats receiving CD4+ T cells from the SS (CD4+ SS→CD247) demonstrated an amplified salt-sensitive hypertension (154±3 mmHg) and albuminuria (184±22 mg/day) compared to blood pressure (140±3 and 145±3 mmHg) and albuminuria (82±9 and 85±8 mg/day) in the CD8+ SS→CD247 or PBS→CD247 groups, respectively. Since the deletion of the p67phox subunit of NADPH oxidase 2 (NOX2) in adoptively transferred splenocytes attenuated Dahl SS hypertension and kidney damage, parallel studies examined the influence of deletion of p67phox in CD4+ (n=14) and CD8+ T cells (n=8). The adoptive transfer of either CD4+ or CD8+ T cells lacking functional NOX2 did not alter the LS or HS blood pressure or albuminuria when compared to the PBS control group. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→CD247 and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells with intact or deficient NOX2. Interestingly, no difference was observed in CD8+ T cells in the kidney and blood of rats receiving CD8+ cells with intact or deficient NOX2, but a significant number of CD4+ T cells were also observed in the SSCD247−/− rats receiving the purified CD8+ T cells. In summary, these studies demonstrate that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS via a NOX2-dependent mechanism while CD8+ T cells do not alter disease phenotypes when compared to PBS control. GRANTS: HL161231, HL166458, Georgia Research Alliance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A bronchiole-on-a-chip and a stretching system for studying human disease model of asthma attack

Asthma is an important public health issue in developed and developing countries, with a prevalence of 300 million worldwide. It is a heterogeneous disease characterized by chronic airway inflammation and hyperresponsiveness. A bronchiole-on-a-chip with a compact stretching system, which is comparable to the small-size incubator and can provide continuous cyclical scratches, is reported here for emulating the physiology of asthma and other types of lung diseases with various breathing rates. In this study, bronchial smooth muscle cells (BSMCs) are cultured on the bronchiole-on-a-chip under cyclic stretching, mimicking the respiratory rates for healthy adults and asthma patients under bronchospasm. With the cyclical cell stretching, the physiology of BSMCs is affected. In an asthma-conditioned bronchial chip, secretions of pro-inflammatory cytokines IL-6 and IL-8 increase significantly from BSMCs compared with the static or healthy condition. In on-chip treatment studies, BSMCs are treated with various concentrations of indacaterol, a long-acting bronchodilator for asthma. The secretion of IL-6 is suppressed for the indacaterol-treated group under asthma conditions. The alignment of stretched BSMCs is also affected by mechanical stretching. Bronchiole-on-a-chip demonstrates the possibility of mimicking the physiological condition of asthma in vitro and a potential drug screening system for lung diseases.

Transcriptional regulation of Hexokinase-2 by BRD4 drives perivascular adipose tissue meta-inflammation in cardiometabolic disease

Aim: To investigate BRD4-related transcriptional programmes in mouse and human models of cardiometabolic disease. Methods: Small arteries (0.1-0.3 mm) dissected from visceral fat biopsies from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were mounted on a pressurized myograph to assess the acute ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated, in the presence or in the absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. A cardiometabolic mouse (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) to test in vivo effect of chronic BRD4 inhibition. ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array, confirmed by ChIP, and characterised by metabolomics, lipidomics and mitochondrial swelling. Results: Endothelial-dependent vasorelaxation and vascular and perivascular TNF-alpha, IL-1beta, IL-6 were altered in cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater than anti-IL-1beta, anti-IL-6 receptor and anti-TNF-alpha. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. Gene expression profiling in PVAT unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validated the findings by demonstrating a glycolytic shift in PVAT under disease conditions. Finally, ex vivo selective inhibition of HK2 rescued vascular dysfunction. Conclusion: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic vascular dysfunction via reversal of the PVAT meta-inflammatory shift, highlighting a novel potential target to fight cardiometabolic pandemics.

Integrated machine learning and multimodal data fusion for patho-phenotypic feature recognition in iPSC models of dilated cardiomyopathy.

Integration of multiple data sources presents a challenge for accurate prediction of molecular patho-phenotypic features in automated analysis of data from human model systems. Here, we applied a machine learning-based data integration to distinguish patho-phenotypic features at the subcellular level for dilated cardiomyopathy (DCM). We employed a human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of a DCM mutation in the sarcomere protein troponin T (TnT), TnT-R141W, compared to isogenic healthy (WT) control iPSC-CMs. We established a multimodal data fusion (MDF)-based analysis to integrate source datasets for Ca2+ transients, force measurements, and contractility recordings. Data were acquired for three additional layer types, single cells, cell monolayers, and 3D spheroid iPSC-CM models. For data analysis, numerical conversion as well as fusion of data from Ca2+ transients, force measurements, and contractility recordings, a non-negative blind deconvolution (NNBD)-based method was applied. Using an XGBoost algorithm, we found a high prediction accuracy for fused single cell, monolayer, and 3D spheroid iPSC-CM models (≥92±0.08 %), as well as for fused Ca2+ transient, beating force, and contractility models (>96±0.04 %). Integrating MDF and XGBoost provides a highly effective analysis tool for prediction of patho-phenotypic features in complex human disease models such as DCM iPSC-CMs.

MiRNA-148a-containing GMSC-derived EVs modulate Treg/Th17 balance via IKKB/NF-κB pathway and treat a rheumatoid arthritis model.

Mesenchymal stem cells (MSCs) have demonstrated potent immunomodulatory properties that have shown promise in the treatment of autoimmune diseases, including rheumatoid arthritis (RA). However, the inherent heterogeneity of MSCs triggered conflicting therapeutic outcomes, raising safety concerns and limiting their clinical application. This study aimed to investigate the potential of extracellular vesicles derived from human gingival mesenchymal stem cells (GMSC-EVs) as a therapeutic strategy for RA. Through in vivo experiments using an experimental RA model, our results demonstrate that GMSC-EVs selectively homed to inflamed joints and recovered Treg and Th17 cell balance, resulting in the reduction of arthritis progression. Our investigations also uncovered miR-148a-3p as a critical contributor to the Treg/Th17 balance modulation via IKKB/NF-κB signaling orchestrated by GMSC-EVs, which was subsequently validated in a model of human xenograft versus host disease (xGvHD). Furthermore, we successfully developed a humanized animal model by utilizing synovial fibroblasts obtained from patients with RA (RASFs). We found that GMSC-EVs impeded the invasiveness of RASFs and minimized cartilage destruction, indicating their potential therapeutic efficacy in the context of patients with RA. Overall, the unique characteristics - including reduced immunogenicity, simplified administration, and inherent ability to target inflamed tissues - position GMSC-EVs as a viable alternative for RA and other autoimmune diseases.

Unveiling new horizons in heart research: the promise of multi-chamber cardiac organoids

Human cardiac and other organoids have recently emerged as a groundbreaking tool for advancing our understanding the developmental biology of human organs. A recent paper from Sasha Mendjan’s laboratory published in the journal Cell on December 7, 2023, reported the generation of multi-chamber cardioids from human pluripotent stem cells, a transformative technology in the field of cardiology. In this short highlight paper, we summarize their findings. Their cardioids remarkably recapitulate the complexity of the human embryonic heart, including tissue architecture, cellular diversity, and functionality providing an excellent in vitro model for investigation of human heart development, disease modeling, precision medicine, and regenerative medicine. Thus, generating cardioids is an important step forward for understanding human heart development and developing potential therapies for heart diseases.