Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

STIM1 ablation impairs exercise-induced physiological cardiac hypertrophy and dysregulates autophagy in mouse hearts.

Cardiac stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry (SOCE), is a known determinant of cardiomyocyte pathological growth in hypertrophic cardiomyopathy. We examined the role of STIM1 and SOCE in response to exercise-dependent physiological hypertrophy. Wild-type (WT) mice subjected to exercise training (WT-Ex) showed a significant increase in exercise capacity and heart weight compared with sedentary (WT-Sed) mice. Moreover, myocytes from WT-Ex hearts displayed an increase in length, but not width, compared with WT-Sed myocytes. Conversely, exercised cardiac-specific STIM1 knock-out mice (cSTIM1KO-Ex), although displaying significant increase in heart weight and cardiac dilation, evidenced no changes in myocyte size and displayed a decreased exercise capacity, impaired cardiac function, and premature death compared with sedentary cardiac-specific STIM1 knock-out mice (cSTIM1KO-Sed). Confocal Ca2+ imaging demonstrated enhanced SOCE in WT-Ex myocytes compared with WT-Sed myocytes with no measurable SOCE detected in cSTIM1KO myocytes. Exercise training induced a significant increase in cardiac phospho-Akt Ser473 in WT mice but not in cSTIM1KO mice. No differences were observed in phosphorylation of mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) in exercised versus sedentary cSTIM1KO mice hearts. cSTIM1KO-Sed mice showed increased basal MAPK phosphorylation compared with WT-Sed that was not altered by exercise training. Finally, histological analysis revealed exercise resulted in increased autophagy in cSTIM1KO but not in WT myocytes. Taken together, our results suggest that adaptive cardiac hypertrophy in response to exercise training involves STIM1-mediated SOCE. Our results demonstrate that STIM1 is involved in and essential for the myocyte longitudinal growth and mTOR activation in response to endurance exercise training.NEW & NOTEWORTHY Store-operated Ca2+ entry (SOCE) has been implicated in pathological cardiac hypertrophy; however, its role in physiological hypertrophy is unknown. Here we report that SOCE is also essential for physiological cardiac hypertrophy and functional adaptations in response to endurance exercise. These adaptations were associated with activation of AKT/mTOR pathway and curtailed cardiac autophagy and degeneration. Thus, SOCE is a common mechanism and an important bifurcation point for signaling paths involved in physiological and pathological hypertrophy.

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform.

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.

Histologic, viral, and molecular correlates of heart disease in fatal COVID-19.

Cardiac manifestations are common in severe COVID-19. This study compared the histologic, viral, and molecular findings in cardiac tissue in fatal COVID-19 (n = 11) and controls (n = 11). In situ hybridization (SARS-CoV2 RNA) and immunohistochemistry for viral proteins and the host response were quantified for the samples and compared with qRTPCR and Western blot data. Control hearts showed a high resident population of macrophages that had variable ACE2 expression. Cardiac ACE2 expression was 10× greater in the heart tissues of cases and controls with obesity or type II diabetes. Multifocal endothelial cell swelling and degeneration, perivascular edema plus microvascular thrombi were unique to the cases. SARS-CoV2 RNA and nucleocapsid protein were rarely detected in situ in any COVID-19 heart. However, in each case abundant SARS-CoV-2 spike protein was evident. Co-expression experiments showed that the spike protein localized mostly to the ACE2+ interstitial macrophages/pericytes that were activated as evidenced by increased IL6 and TNFα expression. Western blots confirmed the presence of the viral spike protein, but not the nucleocapsid protein, in the cardiac homogenates. The intercalated disc proteins connexin 43, the primary cardiac gap junction protein, and NaV1.5, the predominant cardiac sodium channel, each showed marked lateral migration in the myocytes in the cases, which would increase the risk of reentrant arrhythmias. It is concluded that the viral spike protein, endocytosed by macrophages/pericytes, can induce a myocarditis with the possibility of conduction dysfunction due to abnormal localization of key intercalated disc proteins.

Designer Extracellular Vesicles Modulate Pro-Neuronal Cell Responses and Improve Intracranial Retention.

Gene/oligonucleotide therapies have emerged as a promising strategy for the treatment of different neurological conditions. However, current methodologies for the delivery of neurogenic/neurotrophic cargo to brain and nerve tissue are fraught with caveats, including reliance on viral vectors, potential toxicity, and immune/inflammatory responses. Moreover, delivery to the central nervous system is further compounded by the low permeability of the blood brain barrier. Extracellular vesicles (EVs) have emerged as promising delivery vehicles for neurogenic/neurotrophic therapies, overcoming many of the limitations mentioned above. However, the manufacturing processes used for therapeutic EVs remain poorly understood. Here, we conducted a detailed study of the manufacturing process of neurogenic EVs by characterizing the nature of cargo and surface decoration, as well as the transfer dynamics across donor cells, EVs, and recipient cells. Neurogenic EVs loaded with Ascl1, Brn2, and Myt1l (ABM) are found to show enhanced neuron-specific tropism, modulate electrophysiological activity in neuronal cultures, and drive pro-neurogenic conversions/reprogramming. Moreover, murine studies demonstrate that surface decoration with glutamate receptors appears to mediate enhanced EV delivery to the brain. Altogether, the results indicate that ABM-loaded designer EVs can be a promising platform nanotechnology to drive pro-neuronal responses, and that surface functionalization with glutamate receptors can facilitate the deployment of EVs to the brain.

The Cx43 Carboxyl-Terminal Mimetic Peptide αCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner.

The Cx43 carboxyl-terminus (CT) mimetic peptide, αCT1, originally designed to bind to Zonula Occludens 1 (ZO1) and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing (ECIS), a TRITC-dextran Transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular chiral orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell-cell border localization of endothelial VE-cadherin, the tight junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1 binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema.

Vascular endothelial growth factor promotes atrial arrhythmias by inducing acute intercalated disk remodeling.

Atrial fibrillation (AF) is the most common arrhythmia and is associated with inflammation. AF patients have elevated levels of inflammatory cytokines known to promote vascular leak, such as vascular endothelial growth factor A (VEGF). However, the contribution of vascular leak and consequent cardiac edema to the genesis of atrial arrhythmias remains unknown. Previous work suggests that interstitial edema in the heart can acutely promote ventricular arrhythmias by disrupting ventricular myocyte intercalated disk (ID) nanodomains rich in cardiac sodium channels (NaV1.5) and slowing cardiac conduction. Interestingly, similar disruption of ID nanodomains has been identified in atrial samples from AF patients. Therefore, we tested the hypothesis that VEGF-induced vascular leak can acutely increase atrial arrhythmia susceptibility by disrupting ID nanodomains and slowing atrial conduction. Treatment of murine hearts with VEGF (30-60 min, at clinically relevant levels) prolonged the electrocardiographic P wave and increased susceptibility to burst pacing-induced atrial arrhythmias. Optical voltage mapping revealed slower atrial conduction following VEGF treatment (10 ± 0.4 cm/s vs. 21 ± 1 cm/s at baseline, p < 0.05). Transmission electron microscopy revealed increased intermembrane spacing at ID sites adjacent to gap junctions (GJs; 64 ± 9 nm versus 17 ± 1 nm in controls, p < 0.05), as well as sites next to mechanical junctions (MJs; 63 ± 4 nm versus 27 ± 2 nm in controls, p < 0.05) in VEGF-treated hearts relative to controls. Importantly, super-resolution microscopy and quantitative image analysis revealed reorganization of NaV1.5 away from dense clusters localized near GJs and MJs to a more diffuse distribution throughout the ID. Taken together, these data suggest that VEGF can acutely predispose otherwise normal hearts to atrial arrhythmias by dynamically disrupting NaV1.5-rich ID nanodomains and slowing atrial conduction. These data highlight inflammation-induced vascular leak as a potential factor in the development and progression of AF.

Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle.

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease.

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.

microRNA 155 up regulation in the CNS is strongly correlated to Down's syndrome dementia.

This study examined the molecular correlates of Down's dementia. qRTPCR for chromosome 21 microRNAs was correlated with in situ hybridization, immunohistochemistry for microRNA targets, mRNAs located on chromosome 21, and neurofibrillary tangles in human and the Ts65 dn mouse Down's model. qRTPCR for the microRNAs on the triplicated chromosome showed miR-155 dominance in brain tissues (14.3 fold increase, human and 24.2 fold increase, mouse model) that co-expressed with hyperphosphorylated tau protein. miR-155 was not elevated in Alzheimer's disease or neonates with Downs' syndrome. Chromosome 21 genes APP/BA-42, DYRK1a and BACH1 were not correlated to pathologic changes in Down's dementia. Validated CNS targets of miR-155 that were present in controls and Alzheimer's disease but lacking in Down's dementia brains included BACH1, CoREST1, bcl6, BIM, bcl10, cyclin D, and SAPK4. It is concluded that Down's dementia strongly correlated with overexpression of chromosome 21 microRNA 155 with concomitant reduction of multiple CNS-functional targets. This study highlights the need for anatomic pathologists to determine the specific and diverse pathways cells may take to form neurofibrillary tangles in the different dementias.

Increased expression of importin-ß, exportin-5 and nuclear transportable proteins in Alzheimer's disease aids anatomic pathologists in its diagnosis.

Understanding the metabolic profile of neurons with the hyperphosphorylated tau protein characteristic of Alzheimer's disease is essential to unraveling new potential therapies and diagnostics for the surgical pathologist. We stratified 75 brain tissues from Alzheimer's disease into hyperphosphorylated tau positive or negative and did co-expression analyses and qRTPCR for importin-ß and exportin-5 plus several bcl2 family members and compared the data to controls, Down's dementia and Parkinson's disease. There was a significant increase in the expression of importin-ß and exportin-5 in Alzheimer's disease relative to the three other categories (each p value<0.0001) where each protein co-localized with hyperphosphorylated tau. Both apoptotic and anti-apoptotic proteins were each significantly increased in Alzheimer's disease relative to the three other groups. Neurons with hyperphosphorylated tau in Alzheimer's disease have the profile of metabolically active cells including increased exportin-5 and importin-ß mRNA and proteins which indicates that immunohistochemistry testing of these proteins may aid the surgical pathologist in making a definitive diagnosis.

Diagnostic pathology of Alzheimer's disease from routine microscopy to immunohistochemistry and experimental correlations.

The absence of any histologic correlate for Alzheimer's disease despite its commonness and severe clinical sequelae may offers clues to its etiology. Recent evidence strongly suggests that the central event of this disease is the hyperphosphorylation of neuronal tau protein and not the beta amyloid precipitates. In each case, essential and soluble neuronal proteins derivatives form insoluble aggregates that can readily be detected by immunohistochemistry using antibodies specific for the misfolded proteins. Immunohistochemistry also demonstrates that neurons with hyperphosphorylated tau protein are viable. Experimental evidence using neuronal cell cultures suggests that the affected neurons in Alzheimer's disease may have undergone molecular changes that include accumulation of anti-apopotic proteins MCL1 and cFLIP that do not allow the cell to undergo programmed cell death but, rather, to "immortalize" and thus accumulate hyperphosphorylated tau protein in the neuronal cell body and beta amyloid in downstream dendrites. We describe a simplified protocol to demonstrate such changes based on tagged LNA modified microRNA/antimicroRNA oligomers and cell cultures. Co-expression showed that the tagged antimiR-512 strongly localized with the markedly up-regulated proteins MCL1 and cFLIP with concomitant accumulation of hyperphosphorylated tau protein. The data underscore to the anatomic pathologist that the diagnosis of Alzheimer's disease is best accomplished by simple immunohistochemistry tests correlated to the clinical history and the key role pathologists can play in understanding the cause of the disease.

Modulation of PD-L1 and CD8 Activity in Idiopathic and Infectious Chronic Inflammatory Conditions.

Programmed death-ligand 1 (PD-L1) can reduce the immune response by inhibiting CD8 T-cell proliferation and cytotoxic activity. We studied a series of human viral (molloscum, human papillomavirus, herpes simplex, cytomegalovirus, Epstein-Barr virus, smallpox) and bacterial infections (Helicobacter pylori) for the in situ expression of PD-L1, mononuclear cell infiltration, and CD8 activity and compared this to noninfectious idiopathic inflammatory conditions to better define which immune responses may be more highly correlated with an infectious agent. Each viral and bacterial infection showed an increased PD-L1 expression that was most prominent in the mononuclear cell/CD8+ infiltrate surrounding the infection. However, the CD8 cells were mostly quiescent as evidenced by the low Ki67 index and minimal granzyme expression. Using a melanoma mouse model, acute reovirus infection increased PD-L1 expression, but decreased CD8 cytotoxic activity and Treg (FOXP3) cell numbers. In comparison, idiopathic noninfectious chronic inflammatory processes including lichen sclerosis, eczema, Sjogren's disease, and ulcerative colitis showed a comparable strong PD-L1 expression in the mononuclear cell infiltrates but much greater Treg infiltration. However, this strong immunosuppressor profile was ineffective as evidenced by strong CD8 proliferation and granzyme expression. These data suggest that viral and bacterial infections induce a PD-L1 response that, unlike noninfectious chronic inflammatory conditions, dampens the activity of the recruited CD8 cells which, in turn, may enhance the ability of anti-PD-L1 therapy to eliminate the infectious agent.

Enhanced expression of PD L1 in cervical intraepithelial neoplasia and cervical cancers.

Programmed death ligand 1 (PD L1) expression can reduce the immune response in both infectious diseases and cancers. We thus examined PD L1 expression in cervical intraepithelial neoplasias (CINs) and cancers since they each reflect infection by human papillomavirus (HPV). PD L1 protein was not evident by immunohistochemistry in histologically normal cervical epithelia (0/55) even when adjacent to CIN or cancer. PD L1 expression was much increased in CINs (20/21=95%) and cervical squamous cell cancer (56/70=80%) and localized to the dysplastic/neoplastic squamous cells and mononuclear cells, respectively. There was also a significant increase (each P<0.001) in PD L1 detection in mononuclear cells when comparing cervical squamous cell cancers to endometrial (22/115=19%) and ovarian adenocarcinomas (5/40=13%). Co-expression analyses showed that the primary inflammatory cell that contained PD L1 was the CD8+ lymphocyte that strongly concentrated around the dysplastic CIN cells and nests of invasive squamous cancer cells. These data show that PD L1 is a solid biomarker of productive HPV infection of the cervix and that it is significantly upregulated in both the carcinoma and surrounding inflammatory cells in cervical cancer when compared with other gynecologic malignancies. This suggests that anti-PD L1 therapy may have a role in the treatment of cervical cancer.

Reduced miR-512 and the Elevated Expression of Its Targets cFLIP and MCL1 Localize to Neurons With Hyperphosphorylated Tau Protein in Alzheimer Disease.

The cause for the neurofibrillary tangles and plaques in Alzheimer disease likely relates to an abnormal accumulation of their key components, which include ß-amyloid and hyperphosphorylated tau protein. We segregated Alzheimer brain sections from people with end-stage disease into those with abundant hyperphosphorylated tau protein and those without and compared each to normal brains for global microRNA patterns. A significant reduced expression of several microRNAs, including miR-512, was evident in the Alzheimer brain sections with abundant hyperphosphorylated tau. Immunohistochemistry documented that 2 known targets of microRNA-512, cFLIP and MCL1, were significantly over expressed and each colocalized to neurons with the abnormal tau protein. Analysis for apoptosis including activated caspase-3, increased caspase-4 and caspase-8, apoptosis initiating factor, APAF-1 activity, and the TUNEL assay was negative in the areas where neurons showed hyperphosphorylated tau. MCM2 expression, a marker of neuroprogenitor cells, was significantly reduced in the Alzheimer sections that contained the hyperphosphorylated tau. These results suggest that a basic defect in Alzheimer disease may be the reduced microRNA-driven increased expression of proteins that may alter the apoptotic/antiapoptotic balance of neurons. This, in turn, could lead to the accumulation of key Alzheimer proteins such as hyperphosphorylated tau that ultimately prevent normal neuronal function and lead to disease symptomatology.