Biology subjects

Ozkan, A.

Publications and source records attributed to Ozkan, A..

9 recordsLinked to original sources

bioRxiv2025

Endothelial CLK2 as a therapeutic target for acute radiation syndrome

Damage to the vascular endothelium is a major contributor to acute radiation injury in multiple organs that underlies acute radiation syndrome (ARS), yet there are no FDA-approved radiation countermeasures targeting endothelial cells. Use of kinome-scale CRISPR screens performed in cultured human vascular endothelial cells isolated from different organs identified CLK2 as a potential radioprotective target. Pharmacological inhibition of CLK2 using TG003 and Cirtuvivint protected these endothelial cells against radiation injury and reversed its effects across the transcriptome and phospho-proteome. Human Organ Chip models of human intestine and lung that contain organ-specific epithelium and microvascular endothelium faithfully replicated clinical features of ARS when exposed to radiation, which were prevented when treated with CLK2 inhibitors. Thus, CLK2 inhibitors may represent a new class of radiation countermeasure drugs that can protect multiple organs against radiation-induced toxicities in patients with ARS.

molecular biology↗

bioRxiv2024

A Novel Mouse Model Demonstrates In Vivo Replenishment of Central Nervous System Pericytes After Successful Acute Ablation

Central nervous system (CNS) pericytes play crucial roles in vascular development and blood-brain barrier maturation during prenatal development, as well as in regulating cerebral blood flow in adults. They have also been implicated in the pathogenesis of numerous neurological disorders. However, the behavior of pericytes in the adult brain after injury remains poorly understood, partly due to limitations in existing pericyte ablation models. To investigate pericyte responses following acute ablation, we developed a tamoxifen-inducible pericyte ablation model by crossing PDGFR{beta}-P2A-CreERT2 and Rosa26-DTA176 transgenic mouse lines. Using this model, we studied the effects of different tamoxifen doses and conducted histological examinations 15 and 60 days post-injection to assess both short- and long-term impacts of pericyte ablation. Our results demonstrate that a low dose of tamoxifen effectively ablates CNS pericytes in mice without reducing survival or causing significant systemic side effects, such as weight loss. Additionally, we found that the extent of pericyte depletion varies between the cortex and the spinal cords gray and white matter regions. Importantly, both pericyte coverage and numbers increased in the weeks following acute ablation, indicating the regenerative capacity of CNS pericytes in vivo. This model offers a valuable tool for future studies on the role of pericytes in neurological disorders, overcoming the limitations of constitutive pericyte ablation models.

neuroscience↗

bioRxiv2024

Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors

Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry in vivo, or for optimally informative disease modeling and/or therapeutic screening in vitro, it is important to reproduce the type or subtype of neurons involved. No such appropriate in vitro models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor Sox6 is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that Sox6 suppresses a latent neurogenic program by repressing proneural Neurog2 expression by progenitors. We FACS-purify these progenitors from postnatal mouse cortex and establish a culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating Neurog2, Fezf2, while antagonizing Olig2 with VP16:Olig2). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons in vivo. They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed Neurog2-driven differentiation. Neurons induced by Neurog2 instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from cortical progenitors sets a precedent and foundation for in vitro mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair.

neuroscience↗

bioRxiv2023

Concerted epithelial and stromal changes during progression of Barrett's Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis

Esophageal adenocarcinoma arises from Barretts esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barretts esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/544265v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@64c375org.highwire.dtl.DTLVardef@15c6ef2org.highwire.dtl.DTLVardef@177dee0org.highwire.dtl.DTLVardef@c945e7_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractTo obtain a comprehensive picture of the coordinated changes in epithelial, stromal and immune compartments during development of Barretts-associated esophageal adenocarcinoma, patient-matched samples corresponding to various phases of disease progression were collected from 12 patients, each of which had at a given time point lesions at multiple stages progression (matched-normal, metaplasia, dysplasia, and carcinoma). Matched "normal" gastric tissues were also collected. These sample were analyzed by single cell RNA-sequencing (scRNAseq) for single-cell resolution transcriptomics and Copy Number Variant (CNV), by proteomics for extracellular matrix (ECM) proteins, by Atomic Force Microscopy (AFM for tissue stiffness and by CODEX spatial proteomics imaging The integrative multi-omics analysis exposed drastic alterations in cell type composition and shifts in cell states in all three compartments. A large subpopulation of fibroblasts absent in the normal esophagus and characteristic of dysplasia and adenocarcinoma sample, that based on markers would indeed be considered cancer associated fibroblasts (CAF), appeared already in the metaplastic phase. This fibroblast subpopulation had transcriptomes virtually indistinguishable with fibroblasts of the cancer free gastric epithelium in these patients C_FIG

cancer biology↗

bioRxiv2022

Cirrhosis and Inflammation Regulates CYP3A4 Mediated Chemoresistance in Vascularized Hepatocellular Carcinoma-on-a-chip

Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient-specific treatment strategies. In this work, we created novel three-dimensional vascularized HCC-on-a-chips (HCCoC), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness. HCC inflammation was controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450-3A4 (CYP3A4) was investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allowed for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). We showed that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increase vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, we show that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy-associated endothelial porosity increase. The HCCoCs were shown to have utility in uncovering the impact of the tumor microenvironment (TME) during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies. Statement of SignificanceRegulation of drug metabolism during the cancer progression of hepatocellular carcinoma (HCC) can be influential to develop personalized treatment strategies. We created novel vascularized hepatocellular carcinoma-chip (HCCoC) composed of tunable collagen and four main liver-specific cell lines to be used as a preclinical tool. In this model, we found cancer evolution states such as inflammation and cirrhosis increases vascular permeability progressively as a result of increased inflammatory cytokines. Furthermore, delivery of doxorubicin only with embolization improved treatment efficacy by decreasing CYP3A4 activity, which can modulate treatment outcome. Overall, we found different disease states can be influential on CYP3A4, thus its targeting can improve HCC treatment outcome.

bioengineering↗

bioRxiv2021

Individual variability in the human connectome maintains selective cross-modal consistency and shares microstructural signatures

Individuals are different in their behavioural responses and cognitive abilities. Neural underpinnings of individual differences are largely unknown. Here, by using multimodal imaging data including diffusion MRI, functional MRI and MEG, we show the consistency of interindividual variation of connectivity across modalities. We demonstrated that regional differences in individual variability of structural and functional connectomes is characterized by higher variability in association cortices and lower variability in sensory and visual cortices. This pattern is consistent across all modalities at varying degrees as shown by significant alignment between functional and structural connectome variabilities at several clusters of brain regions. Variability in connectivity is associated with cortical myelin content and microstructural properties of connections. Our findings contribute to understanding of individual differences in functional and structural organization of brain and facilitate fingerprinting applications.

neuroscience↗

bioRxiv2021

Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP3A4 Metabolic Activity

Variations in tumor biology from patient to patient combined with the low overall survival rate of hepatocellular carcinoma (HCC) present significant clinical challenges. During the progression of chronic liver diseases from inflammation to the development of HCC, microenvironmental properties, including tissue stiffness and oxygen concentration, change over time. This can potentially impact drug metabolism and subsequent therapy response to commonly utilized therapeutics, such as doxorubicin, multi-kinase inhibitors (e.g., sorafenib), and other drugs, including immunotherapies. In this study, we utilized four common HCC cell lines embedded in 3D collagen type-I gels of varying stiffnesses to mimic normal and cirrhotic livers with environmental oxygen regulation to quantify the impact of these microenvironmental factors on HCC chemoresistance. In general, we found that HCC cells with higher baseline levels of cytochrome p450-3A4 (CYP3A4) enzyme expression, HepG2 and C3Asub28, exhibited a cirrhosis-dependent increase in doxorubicin chemoresistance. Under the same conditions, HCC cell lines with lower CYP3A4 expression, HuH-7 and Hep3B2, showed a decrease in doxorubicin chemoresistance in response to an increase in microenvironmental stiffness. This differential therapeutic response was correlated with the regulation of CYP3A4 expression levels under the influence of stiffness and oxygen variation. In all tested HCC cell lines, the addition of sorafenib lowered the required doxorubicin dose to induce significant levels of cell death, demonstrating its potential to help reduce systemic doxorubicin toxicity when used in combination. These results suggest that patient-specific tumor microenvironmental factors, including tissue stiffness, hypoxia, and CYP3A4 activity levels, may need to be considered for more effective use of chemotherapeutics in HCC patients.

cancer biology↗

bioRxiv2020

An evolutionarily acquired microRNA shapes development of mammalian cortical projections

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are co-expressed across multiple projection neuron subtypes. Here, we discover seventeen CSMN-enriched microRNAs (miRNAs), fifteen of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is the first demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior. Significance StatementThe mammalian central nervous system contains unique projections from the cerebral cortex thought to underpin complex motor and cognitive skills, including the corticospinal tract and corpus callosum. The neurons giving rise to these projections - corticospinal and callosal projection neurons - develop from the same progenitors, but acquire strikingly different fates. The broad evolutionary conservation of known genes controlling cortical projection neuron fates raises the question of how the more narrowly conserved corticospinal and callosal projections evolved. We identify a microRNA cluster selectively expressed by corticospinal projection neurons and exclusive to placental mammals. One of these microRNAs promotes corticospinal fate via regulation of the callosal gene LMO4, suggesting a mechanism whereby microRNA regulation during development promotes evolution of neuronal diversity.

neuroscience↗

bioRxiv2020

Cortical source imaging of resting-state MEG with a high resolution atlas: An evaluation of methods

Non-invasive functional neuroimaging of the human brain at rest can give crucial insight into the mechanisms that underpin healthy cognition and neurological disorders. Magnetoencephalography (MEG) measures extracranial magnetic fields originating from neuronal activity with very high temporal resolution, but requires source reconstruction to make neuroanatomical inferences from these signals. Many source reconstruction algorithms for task-based MEG data are available. However, no consensus yet exists on the optimum algorithm for resting-state data. Here, we evaluated the performance of six commonly-used source reconstruction algorithms based on minimum-norm and beamforming estimates. In the context of human resting-state MEG, we compared the algorithms using quantitative metrics, including resolution properties of inverse solutions and explained variance in sensor-level data. Next, we proposed a data-driven approach to reduce the atlas from the Human Connectome Projects multimodal parcellation of the human cortex. This procedure produced a reduced cortical atlas with 250 regions, optimized to match the spatial resolution and the rank of MEG data from the current generation of MEG scanners. For both voxel-wise and parcellated source reconstructions, we showed that the eLORETA inverse algorithm had zero localization error, high spatial resolution, and superior performance in predicting sensor-level activity. Our comprehensive comparisons and recommandations can serve as a guide for choosing appropriate methodologies in future studies of resting-state MEG.

neuroscience↗