Ehmt2 inactivation in pancreatic epithelial cells shapes the transcriptional landscape and inflammation response of the whole pancreas.

Introduction: The Euchromatic Histone Methyl Transferase Protein 2 (EHMT2), also known as G9a, deposits transcriptionally repressive chromatin marks that play pivotal roles in the maturation and homeostasis of multiple organs. Recently, we have shown that Ehmt2 inactivation in the mouse pancreas alters growth and immune gene expression networks, antagonizing Kras-mediated pancreatic cancer initiation and promotion. Here, we elucidate the essential role of Ehmt2 in maintaining a transcriptional landscape that protects organs from inflammation. Methods: Comparative RNA-seq studies between normal postnatal and young adult pancreatic tissue from Ehmt2 conditional knockout animals (Ehmt2 fl/fl ) targeted to the exocrine pancreatic epithelial cells (Pdx1-Cre and P48 Cre/+ ), reveal alterations in gene expression networks in the whole organ related to injury-inflammation-repair, suggesting an increased predisposition to damage. Thus, we induced an inflammation repair response in the Ehmt2 fl/fl pancreas and used a data science-based approach to integrate RNA-seq-derived pathways and networks, deconvolution digital cytology, and spatial transcriptomics. We also analyzed the tissue response to damage at the morphological, biochemical, and molecular pathology levels. Results and discussion: The Ehmt2 fl/fl pancreas displays an enhanced injury-inflammation-repair response, offering insights into fundamental molecular and cellular mechanisms involved in this process. More importantly, these data show that conditional Ehmt2 inactivation in exocrine cells reprograms the local environment to recruit mesenchymal and immunological cells needed to mount an increased inflammatory response. Mechanistically, this response is an enhanced injury-inflammation-repair reaction with a small contribution of specific Ehmt2-regulated transcripts. Thus, this new knowledge extends the mechanisms underlying the role of the Ehmt2-mediated pathway in suppressing pancreatic cancer initiation and modulating inflammatory pancreatic diseases.

Transcriptional Profiling Underscores the Role of Preprocurement Allograft Metabolism and Innate Immune Status on Outcomes in Human Liver Transplantation.

Objective: The adverse effects of ischemia-reperfusion injury (IRI) remain a principal barrier to a successful outcome after lifesaving orthotopic liver transplantation (OLT). Gene expression during different phases of IRI is dynamic and modified by individual exposures, making it attractive for identifying potential therapeutic targets for improving the number of suitable organs for transplantation and patient outcomes. However, data remain limited on the functional landscape of gene expression during liver graft IRI, spanning procurement to reperfusion and recovery. Therefore, we sought to characterize transcriptomic profiles of IRI during multiple phases in human OLT. Methods: We conducted clinical data analyses, histologic evaluation, and RNA sequencing of 17 consecutive human primary OLT. We performed liver allograft biopsies at 4 time points: baseline (B, before donor cross-clamp), at the end of cold ischemia (CI), during early reperfusion (ER, after revascularization), and during late reperfusion (LR). Data were generated and then recipients grouped by post-OLT outcomes categories: immediate allograft function (IAF; n = 11) versus early allograft dysfunction (EAD; n = 6) groups. Results: We observed that CI (vs B) modified a transcriptomic landscape enriched for a metabolic and immune process. Expression levels of hallmark inflammatory response genes were higher transitioning from CI to ER and decreased from ER to LR. IAF group predominantly showed higher bile and fatty acid metabolism activity during LR compared with EAD group, while EAD group maintained more immunomodulatory activities. Throughout all time points, EAD specimens exhibited decreased metabolic activity in both bile and fatty acid pathways. Conclusions: We report transcriptomic profiles of human liver allograft IRI from prepreservation in the donor to posttransplantation in the recipient. Immunomodulatory and metabolic landscapes across ER and LR phases were different between IAF and EAD allografts. Our study also highlights marker genes for these biological processes that we plan to explore as novel therapeutic targets or surrogate markers for severe allograft injury in clinical OLT.

Type I interferon signaling promotes radioresistance in head and neck cancer.

Despite the promise of concurrent radiotherapy (RT) and immunotherapy in head and neck cancer (HNC), multiple randomized trials of this combination have had disappointing results. To evaluate potential immunologic mechanisms of RT resistance, we compared pre-treatment HNCs that developed RT resistance to a matched cohort that achieved curative status. Gene set enrichment analysis demonstrated that a pre-treatment pro-immunogenic tumor microenvironment (TME), including type II interferon [interferon gamma (IFNγ)] and tumor necrosis factor alpha (TNFα) signaling, predicted cure while type I interferon [interferon alpha (IFNα)] enrichment was associated with an immunosuppressive TME found in tumors that went on to recur. We then used immune deconvolution of RNA sequencing datasets to evaluate immunologic cell subset enrichment. This identified M2 macrophage signaling associated with type I IFN pathway expression in RT-recurrent disease. To further dissect mechanism, we then evaluated differential gene expression between pre-treatment and RT-resistant HNCs from sampled from the same patients at the same anatomical location in the oral cavity. Here, recurrent samples exhibited upregulation of type I IFN-stimulated genes (ISGs) including members of the IFN-induced protein with tetratricopeptide repeats (IFIT) and IFN-induced transmembrane (IFITM) gene families. While several ISGs were upregulated in each recurrent cancer, IFIT2 was significantly upregulated in all recurrent tumors when compared with the matched pre-RT specimens. Based on these observations, we hypothesized sustained type I IFN signaling through ISGs, such as IFIT2, may suppress the intra-tumoral immune response thereby promoting radiation resistance.

Changes in Daily Apparent Diffusion Coefficient on Fully Quantitative Magnetic Resonance Imaging Correlate With Established Genomic Pathways of Radiation Sensitivity and Reveal Novel Biologic Associations.

PURPOSE: Changes in quantitative magnetic resonance imaging (qMRI) are frequently observed during chemotherapy or radiation therapy (RT). It is hypothesized that qMRI features are reflective of underlying tissue responses. It's unknown what underlying genomic characteristics underly qMRI changes. We hypothesized that qMRI changes may correlate with DNA damage response (DDR) capacity within human tumors. Therefore, we designed the current study to correlate qMRI changes from daily RT treatment with underlying tumor transcriptomic profiles. METHODS AND MATERIALS: Study participants were prospectively enrolled (National Clinical Trial 03500081). RNA expression levels for 757 genes from pretreatment biopsies were obtained using a custom panel that included signatures of radiation sensitivity and DDR. Daily qMRI data were obtained from a 1.5 Tesla MR linear accelerator. Using these images, d-slow, d-star, perfusion, and apparent diffusion coefficient-mean values in tumors were plotted per-fraction, over time, and associated with genomic pathways. RESULTS: A total of 1022 qMRIs were obtained from 39 patients and both genomic data and qMRI data from 27 total patients. For 20 of those patients, we also generated normal tissue transcriptomic data. Radio sensitivity index values most closely associated with tissue of origin. Multiple genomic pathways including DNA repair, peroxisome, late estrogen receptor responses, KRAS signaling, and UV response were significantly associated with qMRI feature changes (P < .001). CONCLUSIONS: Genomic pathway associations across metabolic, RT sensitivity, and DDR pathways indicate common tumor biology that may correlate with qMRI changes during a course of treatment. Such data provide hypothesis-generating novel mechanistic insight into the biologic meaning of qMRI changes during treatment and enable optimal selection of imaging biomarkers for biologically MR-guided RT.

EHMT2 Inactivation in Pancreatic Epithelial Cells Shapes the Transcriptional Landscape and Inflammation Response of the Whole Pancreas.

The Euchromatic Histone Methyl Transferase Protein 2 (EHMT2), also known as G9a, deposits transcriptionally repressive chromatin marks that play pivotal roles in the maturation and homeostasis of multiple organs. Recently, we have shown that EHMT2 inactivation alters growth and immune gene expression networks, antagonizing KRAS-mediated pancreatic cancer initiation and promotion. Here, we elucidate the essential role of EHMT2 in maintaining a transcriptional landscape that protects organs from inflammation. Comparative RNA-seq studies between normal postnatal and young adult pancreatic tissue from EHMT2 conditional knockout animals ( EHMT2 fl/fl ) targeted to the exocrine pancreatic epithelial cells ( Pdx1-Cre and P48 Cre/+ ), reveal alterations in gene expression networks in the whole organ related to injury-inflammation-repair, suggesting an increased predisposition to damage. Thus, we induced an inflammation repair response in the EHMT2 fl/fl pancreas and used a data science-based approach to integrate RNA-seq-derived pathways and networks, deconvolution digital cytology, and spatial transcriptomics. We also analyzed the tissue response to damage at the morphological, biochemical, and molecular pathology levels. The EHMT2 fl/fl pancreas displays an enhanced injury-inflammation-repair response, offering insights into fundamental molecular and cellular mechanisms involved in this process. More importantly, these data show that conditional EHMT2 inactivation in exocrine cells reprograms the local environment to recruit mesenchymal and immunological cells needed to mount an increased inflammatory response. Mechanistically, this response is an enhanced injury-inflammation-repair reaction with a small contribution of specific EHMT2-regulated transcripts. Thus, this new knowledge extends the mechanisms underlying the role of the EHMT2-mediated pathway in suppressing pancreatic cancer initiation and modulating inflammatory pancreatic diseases.

Verteporfin induces lipid peroxidation and ferroptosis in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has extremely poor prognosis, with a 5-year survival rate of approximately 11 %. Yes-associated protein (YAP) is a major downstream effector of the Hippo-YAP pathway and plays a pivotal role in regulation of cell proliferation and organ regeneration and tumorigenesis. Activation of YAP signaling has been associated with PDAC progression and drug resistance. Verteporfin (VP) is a photosensitizer used for photodynamic therapy and previous work showed that it can function as a YAP inhibitor. The efficacy of VP on human cancer are being tested in several trials. In this study, we examined the effect of VP on reactive oxygen species (ROS) and lipid peroxidation in pancreatic cancer cells, by using fluorescent molecular probes and by measuring the levels of malondialdehyde, a metabolic byproduct and marker of lipid peroxidation. We found that VP causes rapid increase of both overall ROS and lipid peroxide levels, independent of light activation. These effects were not dependent on YAP, as knockdown of YAP did not cause ROS or lipid peroxidation or enhance VP-induced ROS production. Temoporfin, another photodynamic drug, did not show similar activities. In addition, VP treatment led to loss of cell membrane integrity and reduction of viability. Notably, the activity of VP to induce lipid peroxidation was neutralized by ferroptosis inhibitors ferrostatin-1 or liproxstatin-1. VP treatment also reduced the levels of glutathione peroxidase 4 (GPX4), an enzyme that protects against lipid peroxidation. These results indicate that VP can induce lipid peroxidation and ferroptosis in the absence of light activation. Our findings reveal a novel mechanism by which VP inhibits tumor growth and provide insights into development of new therapeutic strategies for the treatment of pancreatic cancer.

Deep computational phenotyping of genomic variants impacting the SET domain of KMT2C reveal molecular mechanisms for their dysfunction.

Introduction: Kleefstra Syndrome type 2 (KLEFS-2) is a genetic, neurodevelopmental disorder characterized by intellectual disability, infantile hypotonia, severe expressive language delay, and characteristic facial appearance, with a spectrum of other distinct clinical manifestations. Pathogenic mutations in the epigenetic modifier type 2 lysine methyltransferase KMT2C have been identified to be causative in KLEFS-2 individuals. Methods: This work reports a translational genomic study that applies a multidimensional computational approach for deep variant phenotyping, combining conventional genomic analyses, advanced protein bioinformatics, computational biophysics, biochemistry, and biostatistics-based modeling. We use standard variant annotation, paralog annotation analyses, molecular mechanics, and molecular dynamics simulations to evaluate damaging scores and provide potential mechanisms underlying KMT2C variant dysfunction. Results: We integrated data derived from the structure and dynamics of KMT2C to classify variants into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). When compared with controls, these variants show values reflecting alterations in molecular fitness in both structure and dynamics. Discussion: We demonstrate that our 3D models for KMT2C variants suggest distinct mechanisms that lead to their imbalance and are not predictable from sequence alone. Thus, the missense variants studied here cause destabilizing effects on KMT2C function by different biophysical and biochemical mechanisms which we adeptly describe. This new knowledge extends our understanding of how variations in the KMT2C gene cause the dysfunction of its methyltransferase enzyme product, thereby bearing significant biomedical relevance for carriers of KLEFS2-associated genomic mutations.

A multi-layered computational structural genomics approach enhances domain-specific interpretation of Kleefstra syndrome variants in EHMT1.

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction.

A Multi-Layered Computational Structural Genomics Approach Enhances Domain-Specific Interpretation of Kleefstra Syndrome Variants in EHMT1.

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretation of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within this domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism of variant dysfunction.

Beyond structural bioinformatics for genomics with dynamics characterization of an expanded KRAS mutational landscape.

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations, which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

Beyond Structural Bioinformatics for Genomics with Dynamics Characterization of an Expanded KRAS Mutational Landscape.

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

The splanchnic mesenchyme is the tissue of origin for pancreatic fibroblasts during homeostasis and tumorigenesis.

Pancreatic cancer is characterized by abundant desmoplasia, a dense stroma composed of extra-cellular and cellular components, with cancer associated fibroblasts (CAFs) being the major cellular component. However, the tissue(s) of origin for CAFs remains controversial. Here we determine the tissue origin of pancreatic CAFs through comprehensive lineage tracing studies in mice. We find that the splanchnic mesenchyme, the fetal cell layer surrounding the endoderm from which the pancreatic epithelium originates, gives rise to the majority of resident fibroblasts in the normal pancreas. In a genetic mouse model of pancreatic cancer, resident fibroblasts expand and constitute the bulk of CAFs. Single cell RNA profiling identifies gene expression signatures that are shared among the fetal splanchnic mesenchyme, adult fibroblasts and CAFs, suggesting a persistent transcriptional program underlies splanchnic lineage differentiation. Together, this study defines the phylogeny of the mesenchymal component of the pancreas and provides insights into pancreatic morphogenesis and tumorigenesis.

Pepsinogen/Proton Pump Co-Expression in Barrett's Esophageal Cells Induces Cancer-Associated Changes.

EDUCATIONAL OBJECTIVE: At the conclusion of this presentation, participants should better understand the carcinogenic potential of pepsin and proton pump expression in Barrett's esophagus. OBJECTIVE: Barrett's esophagus (BE) is a well-known risk factor for esophageal adenocarcinoma (EAC). Gastric H+ /K+ ATPase proton pump and pepsin expression has been demonstrated in some cases of BE; however, the contribution of local pepsin and proton pump expression to carcinogenesis is unknown. In this study, RNA sequencing was used to examine global transcriptomic changes in a BE cell line ectopically expressing pepsinogen and/or gastric H+ /K+ ATPase proton pumps. STUDY DESIGN: In vitro translational. METHODS: BAR-T, a human BE cell line devoid of expression of pepsinogen or proton pumps, was transduced by lentivirus-encoding pepsinogen (PGA5) and/or gastric proton pump subunits (ATP4A, ATP4B). Changes relative to the parental line were assessed by RNA sequencing. RESULTS: Top canonical pathways associated with protein-coding genes differentially expressed in pepsinogen and/or proton pump expressing BAR-T cells included those involved in the tumor microenvironment and epithelial-mesenchymal transition. Top upstream regulators of coding transcripts included TGFB1 and ERBB2, which are associated with the pathogenesis and prognosis of BE and EAC. Top upstream regulators of noncoding transcripts included p300-CBP, I-BET-151, and CD93, which have previously described associations with EAC or carcinogenesis. The top associated disease of both coding and noncoding transcripts was cancer. CONCLUSIONS: These data support the carcinogenic potential of pepsin and proton pump expression in BE and reveal molecular pathways affected by their expression. Further study is warranted to investigate the role of these pathways in carcinogenesis associated with BE. LEVEL OF EVIDENCE: NA Laryngoscope, 133:59-69, 2023.

Deletion of Thioredoxin Reductase Disrupts Redox Homeostasis and Impairs ß-Cell Function.

Reactive oxygen species (ROS) have been implicated as mediators of pancreatic ß-cell damage. While ß-cells are thought to be vulnerable to oxidative damage, we have shown, using inhibitors and acute depletion, that thioredoxin reductase, thioredoxin, and peroxiredoxins are the primary mediators of antioxidant defense in ß-cells. However, the role of this antioxidant cycle in maintaining redox homeostasis and ß-cell survival in vivo remains unclear. Here, we generated mice with a ß-cell specific knockout of thioredoxin reductase 1 (Txnrd1fl/fl; Ins1Cre/+ , ßKO). Despite blunted glucose-stimulated insulin secretion, knockout mice maintain normal whole-body glucose homeostasis. Unlike pancreatic islets with acute Txnrd1 inhibition, ßKO islets do not demonstrate increased sensitivity to ROS. RNA-sequencing analysis revealed that Txnrd1-deficient ß-cells have increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated genes, and altered expression of genes involved in heme and glutathione metabolism, suggesting an adaptive response. Txnrd1-deficient ß-cells also have decreased expression of factors controlling ß-cell function and identity which may explain the mild functional impairment. Together, these results suggest that Txnrd1-knockout ß-cells compensate for loss of this essential antioxidant pathway by increasing expression of Nrf2-regulated antioxidant genes, allowing for protection from excess ROS at the expense of normal ß-cell function and identity.

Structural bioinformatics enhances the interpretation of somatic mutations in KDM6A found in human cancers.

The histone demethylase KDM6A has recently elicited significant attention because its mutations are associated with a rare congenital disorder (Kabuki syndrome) and various types of human cancers. However, distinguishing KDM6A mutations that are deleterious to the enzyme and their underlying mechanisms of dysfunction remain to be fully understood. Here, we report the results from a multi-tiered approach evaluating the impact of 197 KDM6A somatic mutations using information derived from combining conventional genomics data with computational biophysics. This comprehensive approach incorporates multiple scores derived from alterations in protein sequence, structure, and molecular dynamics. Using this method, we classify the KDM6A mutations into 136 damaging variants (69.0%), 32 tolerated variants (16.2%), and 29 variants of uncertain significance (VUS, 14.7%), which is a significant improvement from the previous classification based on the conventional tools (over 40% VUS). We further classify the damaging variants into 15 structural variants (SV), 88 dynamic variants (DV), and 33 structural and dynamic variants (SDV). Comparison with variant scoring methods used in current clinical diagnosis guidelines demonstrates that our approach provides a more comprehensive evaluation of damaging potential and reveals mechanisms of dysfunction. Thus, these results should be taken into consideration for clinical assessment of the damaging potential of each mutation, as they provide hypotheses for experimental validation and critical information for the development of mutant-specific drugs to fight diseases caused by KDM6A dysfunctions.

NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

RhoBTB1 reverses established arterial stiffness in angiotensin II-induced hypertension by promoting actin depolymerization.

Arterial stiffness predicts cardiovascular disease and all-cause mortality, but its treatment remains challenging. Mice treated with angiotensin II (Ang II) develop hypertension, arterial stiffness, vascular dysfunction, and a downregulation of Rho-related BTB domain-containing protein 1 (RhoBTB1) in the vasculature. RhoBTB1 is associated with blood pressure regulation, but its function is poorly understood. We tested the hypothesis that restoring RhoBTB1 can attenuate arterial stiffness, hypertension, and vascular dysfunction in Ang II-treated mice. Genetic complementation of RhoBTB1 in the vasculature was achieved using mice expressing a tamoxifen-inducible, smooth muscle-specific RhoBTB1 transgene. RhoBTB1 restoration efficiently and rapidly alleviated arterial stiffness but not hypertension or vascular dysfunction. Mechanistic studies revealed that RhoBTB1 had no substantial effect on several classical arterial stiffness contributors, such as collagen deposition, elastin content, and vascular smooth muscle remodeling. Instead, Ang II increased actin polymerization in the aorta, which was reversed by RhoBTB1. Changes in the levels of 2 regulators of actin polymerization, cofilin and vasodilator-stimulated phosphoprotein, in response to RhoBTB1 were consistent with an actin depolymerization mechanism. Our study reveals an important function of RhoBTB1, demonstrates its vital role in antagonizing established arterial stiffness, and further supports a functional and mechanistic separation among hypertension, vascular dysfunction, and arterial stiffness.

Microglia Influence Neurofilament Deposition in ALS iPSC-Derived Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here, we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin which exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte-conditioned medium and microglial-conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.

Enhanced interpretation of 935 hotspot and non-hotspot RAS variants using evidence-based structural bioinformatics.

In the current study, we report computational scores for advancing genomic interpretation of disease-associated genomic variation in members of the RAS family of genes. For this purpose, we applied 31 sequence- and 3D structure-based computational scores, chosen by their breadth of biophysical properties. We parametrized our data by assembling a numerically homogenized experimentally-derived dataset, which when use in our calculations reveal that computational scores using 3D structure highly correlate with experimental measures (e.g., GAP-mediated hydrolysis RSpearman = 0.80 and RAF affinity Rspearman = 0.82), while sequence-based scores are discordant with this data. Performing all-against-all comparisons, we applied this parametrized modeling approach to the study of 935 RAS variants from 7 RAS genes, which led us to identify 4 groups of mutations according to distinct biochemical scores within each group. Each group was comprised of hotspot and non-hotspot KRAS variants, indicating that poorly characterized variants could functionally behave like pathogenic mutations. Combining computational scores using dimensionality reduction indicated that changes to local unfolding propensity associate with changes in enzyme activity by genomic variants. Hence, our systematic approach, combining methodologies from both clinical genomics and 3D structural bioinformatics, represents an expansion for interpreting genomic data, provides information of mechanistic value, and that is transferable to other proteins.