Biology subjects

Gongol, B.

Publications and source records attributed to Gongol, B..

5 recordsLinked to original sources

bioRxiv2025

Integration of artificial intelligence and high-content screening enabled identification of drugs for long-term treatment of cerebral cavernous malformation disease

BackgroundAdults and children with cerebral cavernous malformations (CCMs) are at risk of experiencing lifelong complications such as hemorrhagic strokes, neurological deficits, and epileptic seizures. These complications can severely reduce quality of life. At present, there is no safe or effective therapeutic option for the long-term treatment of CCMs. MethodsUsing advanced artificial intelligence (AI) and machine learning models, powered by the Benevolent Platform, we aimed to identify therapeutic drug targets for CCM pathology (e.g., CCM1, CCM2, CCM3). An AI integrative approach utilized various data types from biomedical entities, including diseases, genes, tissues, and biological mechanisms, together with CCM transcriptomic experimental data. High-throughput drug screening of AI-selected FDA-approved medications, analyses of mitochondrial morphology, and studies on pharmacokinetics, pharmacodynamics, and toxicology were conducted in CCM animal models to identify drugs that could potentially be repurposed for the long-term treatment of CCM disease. ResultsAI predicted the AMPK (AMP-activated protein kinase) and mTOR (mammalian target of rapamycin) pathways as potential therapeutic targets that contribute to CCM pathology. High-content screening validation revealed that the FDA-approved drug metformin, which acts as an AMPK agonist and mTOR inhibitor, can reverse changes in cell-cell junction organization and increase KLF4 expression, a marker for CCM, in human CCM endothelial cells in cultured assays. In addition, pharmacodynamic markers of metformin were observed in CCM mouse models (Slco1c1-iCreERT2;Krit1fl/fl;Ptenfl/wt and Slco1c1-iCreERT2;Pdcd10fl/fl) including reduced S6 kinase or ribosomal protein phosphorylation, a marker of decrease mTOR signaling, and increased AMPK phosphorylation, a marker of AMPK activation, that corresponded to reduced lesion burden. Pharmacokinetic and toxicological studies in CCM animal models showed that that metformin penetrates the brain and long-term administration has a favorable safety profile. We also demonstrated that brain endothelial cells in chronic CCM mouse models exhibit increased levels of the inflammatory marker VCAM-1, which is associated with altered mitochondrial phenotypes, as observed by immunofluorescence, MITO-tagging, and electron microscopy analysis. Additionally, we discovered that metformin and a potent AMPK activator, PF-06409577, can reverse mitochondrial phenotypic changes in brain endothelial cells and reduce the elevation of VCAM-1 expression associated with chronic CCM disease. Therefore, metformin can provide cytoprotection and may reverse the CCM endothelial phenotype by activating AMPK. ConclusionsPredictions using AI technology and high-throughput drug screening, combined with pharmacokinetic, pharmacodynamic, and toxicological studies in CCM animal models, identified metformin as a promising drug candidate for repurposing for the long-term treatment of CCM disease. We propose that metformin enhances metabolic adaptation to brain vascular malformations by activating AMPK, which helps reverse mitochondrial fragmentation in brain endothelial cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/693036v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@17bfadforg.highwire.dtl.DTLVardef@936708org.highwire.dtl.DTLVardef@15171ceorg.highwire.dtl.DTLVardef@69029_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2022

Epitranscriptomic Modification of MicroRNA Increases Atherosclerosis Susceptibility

Emerging evidence indicates that oxidative stress causes the hydroxylation of guanine (G) to generate 8-oxo-7,8-dihydro guanosine (8OH-G) in microRNAs (miRs), which induces the guanine-to-uracil (G-to-U) transversion and thus changes the miR targetomes. However, whether and how the 8OH-G-modified miRs are involved in vascular endothelial dysfunction and atherogenesis were unexplored. Using 8OH-G crosslinking immunoprecipitation miR sequencing (8OH-G CLIP-miR-seq), we found that 8OH-G miR-483 were among the most enriched 8OH-G miR species in ECs induced by ox-LDL. Transcriptomic profiling by RNA-seq indicated that the G-to-U transversion of miR-483 altered the original mRNA targeting efficacy and allows 8OH-G miR-483 to recognize new mRNA target sites. A reduced ratio of 8OH-G miR-483 to miR-483 in lung ECs was found in the endothelial-specific miR-483 transgenic (EC-miR-483 Tg) mice. Moreover, reduction of atherosclerosis was significant in EC-miR-483 Tg mice administrated AAV8-PCSK9 and fed an atherogenic diet. In situ miR hybridization revealed an increased 8OH-G miR-483 level in the intima of human atherosclerotic arteries. Collectively, this study demonstrates that the redox burden incurred by cardiovascular risk factors is a culprit of the miR-483 to 8OH-G miR-483 transversion. Such epitranscriptomic modification of miR-483 causes endothelial dysfunction and increases atherosclerosis susceptibility via its targetomes shift.

molecular biology↗

bioRxiv2022

Neuroinflammation plays a critical role in cerebral cavernous malformation disease

BackgroundCerebral Cavernous Malformations (CCMs) are neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect [~]1/200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. MethodsWe use genetic, RNA-seq, histology, flow cytometry and imaging techniques to report the interaction between CCM-endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CALMN interaction) during the pathogenesis of CCMs in the brain tissue. ResultsExpression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrogliosis. CCM reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-seq analysis on RNA isolated from brain endothelial cells (BECs) in chronic Pdcd10BECKO mice (CCM-endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM- endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA caspase-1) in BECs from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-kB activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in elevated number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-kB inhibition may contribute to detrimental side effects. ConclusionsOur study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-kB activity may contribute to detrimental side effects.

neuroscience↗

bioRxiv2021

Non cell-autonomous effect of astrocytes on cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we report that proliferative astrocytes play a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2 and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilizes HIF-1 in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We show that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1 target gene and a known component of the hypoxic program, contributes to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevents progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

neuroscience↗

bioRxiv2019

Pharmacological inhibition of the heart of glass (HEG1)-Krev interaction trapped protein 1 (KRIT1) protein complex increases Kruppel-like Factors 4 and 2 (KLF4/2) expression in endothelial cells

The Kruppel-like Factors 4 and 2 (KLF4/2) are transcription factors and master regulators of endothelial cells (ECs) phenotype and homeostasis. KLF4/2 are important blood-flow-responsive genes within ECs that differentially regulate the expression of factors that confer anti-inflammatory, antithrombotic, and antiproliferative effects in ECs. Genetic inactivation of endothelial KRIT1 (Krev interaction trapped protein 1) or HEG1 (Heart of glass) lead to upregulation of KLF4/2 expression levels. Furthermore, increased expression of thrombomodulin (THBD) and suppression of thrombospondin (THBS1) was ascribed to elevation of KLF4/2 as a result of loss of endothelial KRIT1. Here, we developed a high-throughput screening assay to identify inhibitors of the HEG1-KRIT1 interaction and identified, HEG1-KRIT1 inhibitor 1 (HKi1), as a promising hit inhibitor. The crystal structure of HKi1 bound to the KRIT1 FERM domain confirmed the primary screening results and ultimately led to the identification of a fragment-like inhibitor (HKi3), which occupies the HEG1 pocket producing comparable activity. These findings suggest that these inhibitors block the interaction by competing with the HEG1 for binding to KRIT1 FERM domain. Moreover, our results demonstrate that HKi3 upregulates KLF4/2 gene expression in two types of human ECs. These results reveal that acute pharmacological inhibition of the HEG1-KRIT1 interaction rapidly induces expression of KLF4/2 and their important transcriptional targets thrombomodulin and thrombospondin.

biochemistry↗