Biology subjects

Lopez-Ramirez, M. A.

Publications and source records attributed to Lopez-Ramirez, M. A..

11 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↗

bioRxiv2025

Activated protein C promotes beta-arrestin-2- and c-Src-dependent caveolin-1 (Cav1) phosphorylation and alters Cav1 association with PAR1 and GRK5

G protein-coupled receptors (GPCRs) display bias towards either G proteins or GPCR kinase (GRK)-mediated {beta}-arrestin signaling depending on the agonist stabilized receptor conformation and cellular context. The cellular location of GPCRs particularly within plasma membrane microdomains such as lipid rafts contributes to biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits biased signaling in response to thrombin and activated protein C (APC). APC-induced {beta}-arrestin-2 ({beta}arr2) biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural protein of caveolae and regulates signaling through protein-protein interactions. The mechanisms by which Cav1 contributes to APC/PAR1-induced {beta}arr2 biased signaling is not known. Here we report that APC-activated PAR1 modulates Cav1 phosphorylation via a {beta}arr2- and c-Src-dependent pathway. APC also regulates the dynamics of endogenous PAR1-Cav1 and GRK5-Cav1 co-localization examined by single molecule super-resolution stochastic optical reconstruction microscopy imaging in human cultured endothelial cells. We further demonstrate that GRK5 interacts with Cav1 in intact cells through an N-terminus aromatic-rich consensus Cav1 binding motif. Unlike wildtype GRK5, a GRK5 mutant defective in Cav1 binding localized predominantly to the cytoplasm rather than the plasma membrane and failed to promote {beta}arr2 recruitment to APC-activated PAR1. These studies suggest that Cav1 itself contributes to the regulation of APC-activated PAR1 {beta}arr2 biased signaling likely through multiple mechanisms that may converge on GRK5.

cell biology↗

bioRxiv2025

Persistent Activation of Endothelial Cells is Linked to Thrombosis and Inflammation in Cerebral Cavernous Malformation Disease

AbstractO_ST_ABSBACKGROUNDC_ST_ABSCerebral cavernous malformations (CCM) are neurovascular lesions that affect both children and adults, and morbidity often results from thrombosis, bleeding, and neurological dysfunction. Studies indicate that inflammation-related activation of endothelial cells contributes significantly to the worsening of CCM disease. This suggests that ongoing vascular inflammation and endothelial dysfunction are key factors associated with thrombosis and bleeding in CCM disease. However, the inflammatory mechanisms leading to altered brain endothelial cell function with a high propensity for thrombosis, inflammation, and dysfunction are not fully understood. METHODSMulti-omic analyses was conducted by performing simultaneous high-throughput single-nucleus RNA sequencing (snRNA-seq) and single-nucleus transposase-accessible chromatin sequencing (snATAC-seq) with the 10x Genomics multiome platform in combination with immunofluorescence to study CCM pathogenesis in both female and male mice with CCM (Slco1c1-CreERT2; Pdcd10fl/fl) disease. The analysis was complemented with bulk RNA-seq, bulk ATAC-seq, and ChIP-seq (Chromatin immunoprecipitation sequencing) using an in vitro human CCM model. An AAV-BR1 viral system selectively upregulates the activator protein-1 (AP-1) transcription factor JUNB in brain endothelial cells was used to evaluate its effectiveness in maintaining a persistent activated cell state during the pathogenesis of CCM. RESULTSWe found that epigenetics significantly influences the subtype identity and function of brain endothelial cells within the arteriovenous axis. Through multi-omic analyses, specific regulatory elements and enhancers (cis-Regulatory Elements, cCREs) in mouse brain endothelial cells were identified that influence subtype-specific transcriptional programs and the transcription factors responsible for establishing the various subtypes of brain endothelial cells. Additionally, large-scale epigenomic reprogramming of brain endothelial cell subtypes was observed during the pathogenesis of CCM disease. Among the most significant changes were alterations in the chromatin state of endothelial cells, along with transcriptional processes associated with a persistently activated endothelial cell state, which renders them susceptible to inflammation and thrombosis. The activator AP-1 transcription factor JUNB was identified as a key regulator of the persistently activated endothelial state during chronic neuroinflammation. Moreover, both trans- and cis-regulatory factors conserved between mice and humans were discovered and contribute to the progression of chronic CCM disease. CONCLUSIONSEpigenetics plays a crucial role in determining the transcription patterns and functions of brain arteriovenous endothelial cells. The activator JUNB is identified as a driver of chronic brain vascular inflammation by inducing a persistent activated endothelial cell state from epigenome reprogramming. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/662238v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1297edforg.highwire.dtl.DTLVardef@1488e27org.highwire.dtl.DTLVardef@6cae62org.highwire.dtl.DTLVardef@12cb282_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Silencing KRIT1 Partially Reverses the Effects of Disturbed Flow onThe Endothelial Cell Transcriptome

BackgroundEndothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow sheer stress are primarily mediated by the modulation of transcription factors Kruppel-like factors 2 and 4 (KLF2 and KLF4). Notably, disturbed flow patterns, which are found in vascular areas predisposed to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4, resulting in changes in the transcriptome that exacerbate inflammation and thrombosis. The endothelial CCM complex, comprising KRIT1, CCM2, and CCM3, suppresses the expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that silencing of KRIT1, the central scaffold of the CCM complex, can normalize the atherogenic effects of disturbed flow on the human endothelial transcriptome MethodsBulk RNA sequencing (RNA-seq) was conducted on human umbilical vein endothelial cells (HUVECs) after the expression of KRIT1 was silenced using specific siRNAs. The endothelial cells were exposed to three different conditions for 24 hours: pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), and static conditions (no flow). ResultsWe found that silencing KRIT1 expression in HUVECs restored the expression of the transcription factors KLF2 and KLF4 under oscillatory shear stress. This treatment resulted in a transcriptomic profile similar to that of endothelial cells under pulsatile shear stress. These findings suggest that inhibition of the CCM complex in endothelium plays a vasoprotective role by reactivating a protective gene program to help endothelial cells resist disturbed blood flow. ConclusionsTargeting CCM genes can activate well-known vasoprotective gene programs that enhance endothelial resilience to inflammation, hypoxia, and angiogenesis under disturbed flow conditions, providing a novel pathway for preventing atherosclerosis. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/642862v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@a20119org.highwire.dtl.DTLVardef@1f62f68org.highwire.dtl.DTLVardef@1a26d71org.highwire.dtl.DTLVardef@bf3c82_HPS_FORMAT_FIGEXP M_FIG C_FIG Endothelial cells respond to blood flow by expressing transcription factors such as Kruppel-Like Factors 2 and 4 (KLF2 and KLF4). Under oscillatory shear stress (OS) conditions, characterized by disturbed flow, KLF2 and KLF4 are suppressed in endothelial cells, which is linked to an atheroprone gene program that includes increased inflammation and risk of atherosclerosis (unhealthy endothelium). Our work shows that silencing of endothelial KRIT1 under OS increases expression of KLF2 and KLF4 mRNA levels as well as the expression of a protective gene program associated with resilience to inflammation, hypoxia, and angiogenesis, similar to those observed under pulsatile shear stress conditions (healthy endothelium).

pathology↗

bioRxiv2024

Behavioral impairments are linked to neuroinflammation in mice with Cerebral Cavernous Malformation disease

BackgroundCerebral Cavernous Malformations (CCMs) are neurovascular abnormalities in the central nervous system (CNS) caused by loss of function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3) genes. One of the most common symptoms in CCM patients is associated with motor disability, weakness, seizures, stress, and anxiety, and the extent of the symptom or symptoms may be due to the location of the lesion within the CNS or whether multiple lesions are present. Previous studies have primarily focused on understanding the pathology of CCM using animal models. However, more research has yet to explore the potential impact of CCM lesions on behavioral deficits in animal models, including effects on short-term and long-term memory, motor coordination, and function. MethodsWe used the accelerating RotaRod test to assess motor and coordination deficits. We also used the open field test to assess locomotor activity and pathology-related behavior and Pavlovian fear conditioning to assess short--and long-term memory deficits. Our behavioral studies were complemented by proteomics, histology, immunofluorescence, and imaging techniques. We found that neuroinflammation is crucial in behavioral deficits in male and female mice with neurovascular CCM lesions (Slco1c1-iCreERT2; Pdcd10fl/fl; Pdcd10BECKO). ResultsFunctional behavior tests in male and female Pdcd10BECKO mice revealed that CCM lesions cause sudden motor coordination deficits associated with the manifestation of profound neuroinflammatory lesions. Our findings indicate that maturation of CCM lesions in Pdcd10BECKO mice also experienced a significant change in short- and long-term memory compared to their littermate controls, Pdcd10fl/fl mice. Proteomic experiments reveal that as CCM lesions mature, there is an increase in pathways associated with inflammation, coagulation, and angiogenesis, and a decrease in pathways associated with learning and plasticity. Therefore, our study shows that Pdcd10BECKO mice display a wide range of behavioral deficits due to significant lesion formation in their central nervous system and that signaling pathways associated with neuroinflammation and learning impact behavioral outcomes. ConclusionsOur study found that CCM animal models exhibited behavioral impairments such as decreased motor coordination and amnesia. These impairments were associated with the maturation of CCM lesions that displayed a neuroinflammatory pattern.

animal behavior and cognition↗

bioRxiv2024

Genetic Inactivation of the beta1 adrenergic receptor prevents Cerebral Cavernous Malformations in zebrafish

Propranolol reduces experimental murine cerebral cavernous malformations (CCMs) and prevents embryonic caudal venous plexus (CVP) lesions in zebrafish that follow mosaic inactivation of ccm2. Because morpholino silencing of the {beta}1 adrenergic receptor (adrb1) prevents the embryonic CVP lesion, we proposed that adrb1 plays a role in CCM pathogenesis. Here we report that adrb1-/- zebrafish exhibited 86% fewer CVP lesions and 87% reduction of CCM lesion volume relative to wild type brood mates at 2dpf and 8-10 weeks stage, respectively. Treatment with metoprolol, a {beta}1 selective antagonist, yielded a similar reduction in CCM lesion volume. Adrb1-/- zebrafish embryos exhibited reduced heart rate and contractility and reduced CVP blood flow. Similarly, slowing the heart and eliminating the blood flow in CVP by administration of 2,3-BDM suppressed the CVP lesion. In sum, our findings provide genetic and pharmacological evidence that the therapeutic effect of propranolol on CCM is achieved through {beta}1 receptor antagonism.

developmental 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

Microneedle-mediated intratumoral delivery of anti-CTLA-4 promotes cDC1-dependent eradication of oral squamous cell carcinoma with limited irAEs

Head and neck squamous cell carcinoma (HNSCC) ranks 6th in cancer incidence worldwide and has a five-year survival rate of only 63%. Immunotherapies - principally immune checkpoint inhibitors (ICI), such as anti-PD-1 and anti-CTLA-4 antibodies that restore endogenous antitumor T-cell immunity - offer the greatest promise for HNSCC treatment. Anti-PD-1 has been recently approved for first line treatment of recurrent and metastatic HNSCC; however less than 20% of patients show clinical benefit and durable responses. In addition, the clinical application of ICI has been limited by immune-related adverse events (irAEs) consequent to compromised peripheral immune tolerance. Although irAEs are often reversible, they can become severe, prompting premature therapy termination or becoming life-threatening. To address the irAEs inherent to systemic ICI therapy, we developed a novel, local delivery strategy based upon an array of soluble microneedles (MN). Using our recently reported syngeneic, tobacco-signature murine HNSCC model, we found that both systemic and local-MN anti-CTLA-4 therapy lead to >90% tumor response, which is dependent on CD8 T cells and conventional dendritic cell type 1 (cDC1). However, local-MN delivery limited the distribution of anti-CTLA-4 antibody from areas distal to draining lymphatic basins. Employing Foxp3-GFPDTR transgenic mice to interrogate irAEs in vivo, we found that local-MN delivery of anti-CTLA-4 protects animals from irAEs observed with systemic therapy. Taken together, our findings support the exploration of MN-intratumoral ICI delivery as a viable strategy for HNSCC treatment with reduced irAEs, and the opportunity to target cDC1s as part of multimodal treatment options to boost ICI therapy.

cancer biology↗

bioRxiv2021

The connection between Rap1 and Talin1 in CD4+ T Lymphocytes

Agonist induced increase in integrin affinity for ligands (activation) plays a pivotal role in T cell trafficking and functions. Activation requires Rap1 GTPase-mediated recruitment of talin1 to the integrins in the plasma membrane. Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that serves this function in T cells. In addition, Rap1 directly binds to talin1 to enable integrin activation in platelets. Here, we assessed the relative contributions of the Rap1-talin1 interaction and RIAM and provide a complete accounting of the connections between Rap1 and talin1 that support integrin activation in conventional CD4+ (Tconv) and CD25HiFoxp3+CD4+ regulatory T (Treg) cells. Disruption of both Rap1 binding sites in talin1 (talin1 (R35E,R118E)) causes a partial defect in L{beta}2, 4{beta}1 and 4{beta}7 integrin activation in both Tconv and Treg cells with resulting defects in T cell homing and functions. Over-expression of RIAM bypasses the integrin activation defect in Tconv cells expressing talin1 (R35E,R118E), indicating that RIAM can substitute for Rap1 binding to talin in integrin activation. Conversely, deletion of RIAM in talin1 (R35E,R118E) Tconv cells abrogates activation of L{beta}2, 4{beta}1 and 4{beta}7. RIAM and lamellipodin (LPD) are mammalian members of the MRL protein family; LPD plays a more important role than RIAM in Treg cell integrin activation. Nevertheless, loss of RIAM profoundly exacerbates the defects in Treg cell function caused by the talin1 (R35E,R118E) mutation. Most importantly, deleting both MRL proteins combined with talin1 (R35E,R118E) phenocopies the complete lack of integrin activation observed in Rap1a/b null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable L{beta}2, 4{beta}1 and 4{beta}7 integrin activation in T cells.

cell biology↗

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↗

bioRxiv2018

A Rap1 binding site and lipid-dependent helix in talin F1 domain cooperate in integrin activation

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a negligible contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain contains a previously undetected Rap1 binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that transforms into an amphipathic helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical as charge reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1 binding site and a lipid-dependent amphipathic helix in talin1 F1 cooperate to enable a direct Rap1-talin1 interaction to cause integrin activation.\n\nSummaryThis work reveals that Rap1 GTPases bind directly to talin1 F1 domain and by cooperating with a unique lipid-dependent amphipathic helix in the F1 domain effects talin1-mediated integrin activation.

cell biology↗