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Cerebral small vessel disease (CSVD) has emerged as a common factor driving age-dependent diseases, including stroke and dementia. CSVD-related dementia will affect a growing fraction of the aging population, requiring improved recognition, understanding, and treatments. This review describes evolving criteria and imaging biomarkers for the diagnosis of CSVD-related dementia. We describe diagnostic challenges, particularly in the context of mixed pathologies and the absence of highly effective biomarkers for CSVD-related dementia. We review evidence regarding CSVD as a risk factor for developing neurodegenerative disease and potential mechanisms by which CSVD leads to progressive brain injury. Finally, we summarize recent studies on the effects of major classes of cardiovascular medicines relevant to CSVD-related cognitive impairment. Although many key questions remain, the increased attention to CSVD has resulted in a sharper vision for what will be needed to meet the upcoming challenges imposed by this disease.

The brain is assumed to be hypoactive during cardiac arrest. However, animal models of cardiac and respiratory arrest demonstrate a surge of gamma oscillations and functional connectivity. To investigate whether these preclinical findings translate to humans, we analyzed electroencephalogram and electrocardiogram signals in four comatose dying patients before and after the withdrawal of ventilatory support. Two of the four patients exhibited a rapid and marked surge of gamma power, surge of cross-frequency coupling of gamma waves with slower oscillations, and increased interhemispheric functional and directed connectivity in gamma bands. High-frequency oscillations paralleled the activation of beta/gamma cross-frequency coupling within the somatosensory cortices. Importantly, both patients displayed surges of functional and directed connectivity at multiple frequency bands within the posterior cortical “hot zone,” a region postulated to be critical for conscious processing. This gamma activity was stimulated by global hypoxia and surged further as cardiac conditions deteriorated in the dying patients. These data demonstrate that the surge of gamma power and connectivity observed in animal models of cardiac arrest can be observed in select patients during the process of dying.

Supplementary Figures S1-S7; Supplementary Tables S1-S6.

Purpose: LRP1 (low-density lipoprotein receptor–related protein 1) is a broadly expressed receptor that binds multiple extracellular ligands and participates in protein clearance. It is expressed in numerous cancers, but its role in lung cancer has not been characterized. Here, we investigate the relationship between LRP1 and lung cancer.Experimental Design:LRP1 mRNA levels were determined in lung tumors from several large, multicenter studies. LRP1 protein localization was determined by immunohistochemical analysis of lung tumor microarrays. Normal fibroblasts, fibroblasts treated with the LRP1 inhibitor RAP (receptor-associated protein), and Lrp1 null fibroblasts were cocultured with 3 independent lung cancer cell lines to investigate the role of LRP1 on tumor cell proliferation.Results:LRP1 mRNA levels are significantly decreased in lung tumors relative to nontumorous lung tissue. Lower expression of LRP1 in lung adenocarcinomas correlates with less favorable clinical outcome in a cohort of 439 patients. Immunohistochemical analysis shows that LRP1 is primarily expressed in stromal cells in 94/111 lung cancers, with very little protein found in cancer cells. A growth-suppressive function of mouse embryonic fibroblast (MEF) cells was observed in 3 lung cancer cell lines tested (H460, H2347, and HCC4006 cells); growth suppression was blocked by the LRP1 inhibitor RAP. Lrp1 deletion in fibroblasts reduced the ability of MEF cells to suppress tumor cell mitosis. In a validation set of adenocarcinomas, we confirmed a significant, positive correlation between both LRP1 mRNA and protein levels and favorable clinical outcomes.Conclusions:LRP1 expression is associated with improved lung cancer outcomes. Mechanistically, stromal LRP1 may non–cell autonomously suppress lung tumor cell proliferation. Clin Cancer Res; 17(8); 2426–33. ©2011 AACR.

Indolethylamine N-methyltransferase (INMT) is a transmethylation enzyme that utilizes the methyl donor S-adenosyl-L-methionine to transfer methyl groups to amino groups of small molecule acceptor compounds. INMT is best known for its role in the biosynthesis of N,N-Dimethyltryptamine (DMT), a psychedelic compound found in mammalian brain and other tissues. In mammals, biosynthesis of DMT is thought to occur via the double methylation of tryptamine, where INMT first catalyzes the biosynthesis of N-methyltryptamine (NMT) and then DMT. However, it is unknown whether INMT is necessary for the biosynthesis of endogenous DMT. To test this, we generated a novel INMT-knockout rat model and studied tryptamine methylation using radiometric enzyme assays, thin-layer chromatography, and ultra-high-performance liquid chromatography tandem mass spectrometry. We also studied tryptamine methylation in recombinant rat, rabbit, and human INMT. We report that brain and lung tissues from both wild type and INMT-knockout rats show equal levels of tryptamine-dependent activity, but that the enzymatic products are neither NMT nor DMT. In addition, rat INMT was not sufficient for NMT or DMT biosynthesis. These results suggest an alternative enzymatic pathway for DMT biosynthesis in rats. This work motivates the investigation of novel pathways for endogenous DMT biosynthesis in mammals.

BACKGROUND. Small vessel disease is a prevalent age-related condition linked to increased risk of dementia and stroke. We investigate the most commonly inherited form, CADASIL, caused by cysteine-involving mutations in NOTCH3. Recent studies highlight accumulation of NOTCH3 N-terminal fragmentation product (NTF) in disease. In vitro, NTF is capable of both spontaneous and catecholamine-enhanced cysteine-mediated oligomerization. Despite well-characterized genetic influence on CADASIL, environmental effects, including medication usage, on disease remain unclear. METHODS. We studied effects of assorted electrophilic compounds and drugs on NTF oligomerization by SDS-PAGE and Dynamic Light Scattering. We then examined direct proton pump inhibitor-NTF binding with antibodies designed against proton pump inhibitor-labeled proteins and mass spectrometry. Finally, we used monoclonal NTF antibodies with Proximity Ligation Assay to identify NTF oligomers in 3 CADASIL and 2 age-matched control brains. RESULTS. We identified enhancement of NTF oligomerization by two electrophilic cysteine-modifying compounds, N-ethylmaleimide and iodoacetamide, and an electrophilic compound capable of oxidizing cysteines, ferric chloride. Electrophilic clinical drugs (fenoldopam, omeprazole, tenatoprazole, lansoprazole, and rabeprazole) also promoted oligomerization, and we identified direct omeprazole-NTF and tenatoprazole-NTF complexes. Additionally, we provide novel evidence of NTF multimers in human CADASIL brains. CONCLUSIONS. A broad array of electrophilic chemicals, including clinically relevant drugs, influences oligomerization of a pathological CADASIL protein, providing mechanistic insight into disease protein oligomerization. We posit that environmental influences, which may include usage of electrophilic drugs, may affect CADASIL presentations.

Mutations in NOTCH3 underlie CADASIL, the most common inherited cerebral small vessel disease. Two cleavages of NOTCH3 protein, at Asp80 and Asp121, have been previously described in CADASIL pathological samples. Using monoclonal antibodies developed against a NOTCH3 neo-epitope, we now identify a third cleavage at Asp964 between an Asp-Pro sequence. We characterized the structural requirements for the proteolysis at Asp964 and the vascular distribution of the cleavage event. A proteome-wide analysis was performed to find proteins that interact with the cleavage product. Finally, we investigated the biochemical determinants of this third cleavage event. Cleavage at Asp964 was critically dependent on the proline adjacent to the aspartate residue. In addition, the cleavage product was highly enriched in CADASIL brain tissue and localized to the media of degenerating arteries, where it co-deposited with the two additional NOTCH3 cleavage products. Recombinant NOTCH3 terminating at Asp964 was used to probe protein microarrays, and we identified multiple molecules that bound to the cleaved NOTCH3 more than to uncleaved protein, suggesting that cleavage may alter the local protein interactome within disease-affected blood vessels. The cleavage of purified NOTCH3 protein at Asp964 in vitro was activated by reducing agents and NOTCH3 protein; meanwhile, cleavage was inhibited by specific dicarboxylic acids, as seen with cleavage at Asp80 and Asp121. Overall, we propose homologous redox-driven Asp-Pro cleavages and alterations in protein interactions as potential mechanisms in inherited small vessel disease; similarities in modulation of protein cleavage characteristics may indicate common biochemical modulators of pathological NOTCH3 processing.

Cysteine oxidation states of extracellular proteins participate in functional regulation and in disease pathophysiology. In the most common inherited dementia, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), mutations in NOTCH3 that alter extracellular cysteine number have implicated NOTCH3 cysteine states as potential triggers of cerebral vascular smooth muscle cytopathology. In this report, we describe a novel property of the second EGF-like domain of NOTCH3: its capacity to alter the cysteine redox state of the NOTCH3 ectodomain. Synthetic peptides corresponding to this sequence (NOTCH3 N-terminal fragment 2, NTF2) readily reduce NOTCH3 N-terminal ectodomain polypeptides in a dose- and time-dependent fashion. Furthermore, NTF2 preferentially reduces regional domains of NOTCH3 with the highest intensity against EGF-like domains 12–15. This process requires cysteine residues of NTF2 and is also capable of targeting selected extracellular proteins that include TSP2 and CTSH. CADASIL mutations in NOTCH3 increase susceptibility to NTF2-facilitated reduction and to trans-reduction by NOTCH3 produced in cells. Moreover, NTF2 forms complexes with the NOTCH3 ectodomain, and cleaved NOTCH3 co-localizes with the NOTCH3 ectodomain in cerebral arteries of CADASIL patients. The potential for NTF2 to reduce vascular proteins and the enhanced preference for it to trans-reduce mutant NOTCH3 implicate a role for protein trans-reduction in cerebrovascular pathological states such as CADASIL.

Background and Purpose— Cardiac telemetry is a routine part of inpatient ischemic stroke/transient ischemic attack evaluation to assess for atrial fibrillation (AF). Yet, tools to assist stroke clinicians in the evaluation of the large quantities of telemetry data are limited. The investigators developed a new method to evaluate electrocardiographic signals, electrocardiomatrix, that was applied to stroke unit telemetry data to determine its feasibility, validity, and usefulness. Electrocardiomatrix displays telemetry data in a 3-dimensional matrix that allows for more accurate and less time consuming P-wave analysis. Methods— In this single-center, prospective, observational study conducted in a stroke unit, all telemetry data from ischemic stroke and transient ischemic attack patients were collected (April 2017–January 2018) for examination facilitated by electrocardiomatrix. AF>30 seconds was identified through review of electrocardiomatrix-generated matrices by a nonphysician researcher. Electrocardiomatrix results were compared with the clinical team’s medical record documentation of AF identified through telemetry. A study cardiologist reviewed the standard telemetry associated with all AF episodes identified by electrocardiomatrix and each case of disagreement. Results— Telemetry data (median 46 hours [interquartile range: 22–90]) were analyzed among 265 unique subjects (88% ischemic stroke). Electrocardiomatrix was successfully applied in 260 (98%) of cases. The positive predictive value of electrocardiomatrix compared with the clinical documentation was 86% overall and 100% among the subset with no prior history of AF. For the 5 false-positive and 5 false-negative cases, expert overview disagreed with the clinical documentation and confirmed the electrocardiomatrix-based diagnosis. Conclusions— The application of electrocardiomatrix to stroke unit-acquired telemetry data is feasible and appears to have superior accuracy compared with traditional monitor analysis by noncardiologists.

N,N-dimethyltryptamine (DMT), a psychedelic compound identified endogenously in mammals, is biosynthesized by aromatic-L-amino acid decarboxylase (AADC) and indolethylamine-N-methyltransferase (INMT). Whether DMT is biosynthesized in the mammalian brain is unknown. We investigated brain expression of INMT transcript in rats and humans, co-expression of INMT and AADC mRNA in rat brain and periphery, and brain concentrations of DMT in rats. INMT transcripts were identified in the cerebral cortex, pineal gland, and choroid plexus of both rats and humans via in situ hybridization. Notably, INMT mRNA was colocalized with AADC transcript in rat brain tissues, in contrast to rat peripheral tissues where there existed little overlapping expression of INMT with AADC transcripts. Additionally, extracellular concentrations of DMT in the cerebral cortex of normal behaving rats, with or without the pineal gland, were similar to those of canonical monoamine neurotransmitters including serotonin. A significant increase of DMT levels in the rat visual cortex was observed following induction of experimental cardiac arrest, a finding independent of an intact pineal gland. These results show for the first time that the rat brain is capable of synthesizing and releasing DMT at concentrations comparable to known monoamine neurotransmitters and raise the possibility that this phenomenon may occur similarly in human brains.

White matter injury is a crucial component of human stroke, but it has often been neglected in preclinical studies. Most human stroke is associated with one or more comorbidities, including aging, hypertension, diabetes and metabolic syndrome including hyperlipidemia. The purpose of this review is to examine how age and hypertension impact stroke-induced white matter injury as well as white matter repair in both human stroke and preclinical models. It is essential that comorbidities be examined in preclinical trials as they may impact translatability to the clinic. In addition, understanding how comorbidities impact white matter injury and repair may provide new therapeutic opportunities for patients with those conditions.

Sudden death is an important but underrecognized consequence of stroke. Acute stroke can disturb central control of autonomic function and result in cardiac dysfunction and sudden death. Previous study showed that bilateral common carotid artery ligation (BCCAL) in the spontaneously hypertensive stroke-prone rat strain (SHRSP) is a well-established model for forebrain ischemic sudden death. This study aims to investigate the temporal dynamic changes in electrical activities of the brain and heart and functional interactions between the two vital organs following forebrain ischemia. EEG and ECG signals were simultaneously collected from nine SHRSP and eight Wistar-Kyoto (WKY) rats. RR interval was analyzed to investigate the cardiac response to brain ischemia. EEG power and coherence (CCoh) analysis were conducted to study the cortical response. Corticocardiac coherence (CCCoh) and directional connectivity (CCCon) were analyzed to determine brain-heart connection. Heart rate variability (HRV) was analyzed to evaluate autonomic functionality. BCCAL resulted in 100% mortality in SHRSP within 14 h, whereas no mortality was observed in WKY rats. The functionality of both the brain and the heart were significantly altered in SHRSP compared with WKY rats after BCCAL. SHRSP, but not WKY rats, exhibited intermittent surge of CCCoh, which paralleled the elevated CCCon and reduced HRV, following the onset of ischemia until sudden death. Elevated brain-heart coupling invariably associated with the disruption of the autonomic nervous system and the risk of sudden death. This study may improve our understanding of the mechanism of forebrain ischemia-induced sudden death. NEW & NOTEWORTHY This study demonstrates a marked surge of corticocardiac coupling in rats dying from focal cerebral ischemia, consistent with our earlier data in rats exposed to fatal asphyxia. Since the bidirectional electrical signal coupling (corticocardiac coherence) and communication (corticocardiac connectivity) between the brain and the heart are only identified in dying animals, they could be used as potential biomarkers to predict the risk of sudden death.

SummaryExpression quantitative trait loci (eQTLs) characterize the associations between genetic variation and gene expression to provide insights into tissue-specific gene regulation. Interactive visualization of tissue-specific eQTLs can facilitate our understanding of functional variants relevant to disease-related traits. However, combining the multi-dimensional nature of eQTLs into a concise and informative visualization is challenging. Existing eQTL visualization tools provide useful ways to summarize the unprecedented scale of transcriptomic data but are not necessarily tailored to answer questions about the functional interpretations of trait-associated variants or other variants of interest. We developed FIVEx, an interactive eQTL browser with an intuitive interface tailored to the functional interpretation of associated variants. It features the ability to navigate seamlessly between different data views while providing relevant tissue- and locus-specific information to offer users a better understanding of population-scale multi-tissue transcriptomic profiles. Our implementation of the FIVEx browser on the Gene-Tissue Expression (GTEx) dataset provides important insights for understanding potential tissue-specific regulatory mechanisms underlying trait-associated signals.Availability and implementationA FIVEx instance visualizing GTEx v8 data can be found at https://eqtl.pheweb.org. The FIVEx source code is open source under an MIT license at https://github.com/statgen/fivex.

Omeprazole is the most commonly used proton pump inhibitor (PPI), a class of medications whose therapeutic mechanism of action involves formation of a disulfide linkage to cysteine residues in the H+/K+ ATPase pump on gastric secretory cells. Covalent linkage between the sole sulfur group of omeprazole and selected cysteine residues of the pump protein results in inhibition of acid secretion in the stomach, an effect that ameliorates gastroesophageal reflux and peptic ulcer disease. PPIs, though useful for specific conditions when used transiently, are associated with diverse untoward effects when used long term. The mechanisms underlying these potential off-target effects remain unclear. PPIs may, in fact, interact with non-canonical target proteins (non-pump molecules) resulting in unexpected pathophysiological effects, but few studies describe off-target PPI binding. Here, we describe successful cloning of monoclonal antibodies against protein-bound omeprazole. We developed and used monoclonal antibodies to characterize the protein target range of omeprazole, stability of omeprazole-bound proteins, and the involvement of cysteines in binding of omeprazole to targets. We demonstrate that a wide range of diverse proteins are targeted by omeprazole. Protein complexes, detected by Western blotting, are resistant to heat, detergents, and reducing agents. Reaction of omeprazole occurs with cysteine-free proteins, is not fully inhibited by cysteine alkylation, occurs at neutral pH, and induces protein multimerization. At least two other clinically used PPIs, rabeprazole and tenatoprazole, are capable of binding to proteins in a similar fashion. We conclude that omeprazole binds to multiple proteins and is capable of forming highly stable complexes that are not dependent on disulfide linkages between the drug and protein targets. Further studies made possible by these antibodies may shed light on whether PPI-protein complexes underlie off-target untoward effects of chronic PPI use.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and cerebral amyloid angiopathy (CAA) are two distinct vascular angiopathies that share several similarities in clinical presentation and vascular pathology. Given the clinical and pathologic overlap, the molecular overlap between CADASIL and CAA was explored. CADASIL and CAA protein profiles from recently published proteomics-based and immuno-based studies were compared to investigate the potential for shared disease mechanisms. A comparison of affected proteins in each disease highlighted 19 proteins that are regulated in both CADASIL and CAA. Functional analysis of the shared proteins predicts significant interaction between them and suggests that most enriched proteins play roles in extracellular matrix structure and remodeling. Proposed models to explain the observed enrichment of extracellular matrix proteins include both increased protein secretion and decreased protein turnover by sequestration of chaperones and proteases or formation of stable protein complexes. Single-cell RNA sequencing of vascular cells in mice suggested that the vast majority of the genes accounting for the overlapped proteins between CADASIL and CAA are expressed by fibroblasts. Thus, our current understanding of the molecular profiles of CADASIL and CAA appears to support potential for common mechanisms underlying the two disorders.

A large number of pre-clinical and developmental investigations involve experimental vertebrate animals, of which mice have emerged as a favored organism. Recognition of the differences between humans and mice is essential for assessment of the relevance of animal studies to humans. The primary purpose of this study was to gauge the conservation between human and mouse vascular smooth muscle cell (VSMC) proteins mined from an analysis of the Human Protein Atlas. Two comparison were made: a) immunohistochemistry for 16 proteins in brain, heart, esophagus, bladder, stomach, lung, kidney, and aorta enabled comparison between human and mouse of protein localization in VSMC and non-vascular SMC; and b) multi-species primary protein sequence analysis of an expanded set vascular molecules enabled comparison between VSMC sequences among vertebrate species. In total, three dimensions of diversity were uncovered. First, a significant number of factors show human/mouse differences in cellular expression; these differences occurred in both VSMC and non-vascular SMC in an organ and cell-type dependent fashion. Many markers demonstrated notable cell-to-cell and regional heterogeneity in VSMC of the aorta and non-vascular SMC of the esophagus, bladder, and stomach. Second, species specificity can arise by genetic deletions as exemplified by the human protein adipogenesis regulatory factor (ADIRF), which is not present due to a large sequence gap in mice. Third, we describe significant cross-species protein sequence divergence in selected VSMC proteins which may result in altered orthologue function. In a sample of 346 vascular molecules, 15% demonstrate incomplete vertebrate species gene conservation. Divergence of predicted human/mouse VSMC protein sequences is higher than for endothelial proteins in all species examined. In the future, each of these three cross-species differences could be neutralized using gene manipulation, resulting in improved translational potential of murine experimental models.

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Immunometabolic changes have been shown to be a key factor in determining the immune cell response in disease models. The immunometabolite, itaconate, is produced by aconitate decarboxylase 1 (Acod1) and has been shown to inhibit inflammatory signaling in macrophages. In this study, we explore the role of Acod1 and itaconate in cerebral ischemia/reperfusion injury. We assessed the effect of global Acod1 knockout (Acod1KO, loss of endogenous itaconate) in a transient ischemia/reperfusion occlusion stroke model. Mice received a transient 90-min middle cerebral artery occlusion followed with 24-h of reperfusion. Stroke lesion volume was measured by MRI analysis and brain tissues were collected for mRNA gene expression analysis. Acod1KO mice showed significant increases in lesion volume compared to control mice, however no differences in pro-inflammatory mRNA levels were observed. Cell specific knockout of Acod1 in myeloid cells (LysM-Cre), microglia cells (CX3CR1, Cre-ERT2) and Endothelial cells (Cdh5(PAC), Cre-ERT2) did not reproduce lesion volume changes seen in global Acod1KO, indicating that circulating myeloid cells, resident microglia and endothelial cell populations are not the primary contributors to the observed phenotype. These effects however do not appear to be driven by changes in inflammatory gene regulation. These data suggests that endogenous Acod1 is protective in cerebral ischemia/reperfusion injury.

A variety of conditioning stimuli (e.g. ischemia or hypoxia) can protect against stroke-induced brain injury. While most attention has focused on the effects of conditioning on parenchymal injury, there is considerable evidence that such stimuli also protect the cerebrovasculature, including the blood-brain barrier. This review summarizes the data on the cerebrovascular effects of ischemic/hypoxic pre-, per- and post-conditioning and the mechanisms involved in protection. It also addresses some important questions: Are the cerebrovascular effects of conditioning just secondary to reduced parenchymal injury? How central is endothelial conditioning to overall brain protection? For example, is endothelial conditioning sufficient or necessary for the induction of brain protection against stroke? Is the endothelium crucial as a sensor/transducer of conditioning stimuli?

Introduction: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic cause of cSVD, stroke, and vascular dementia. CADASIL is characterized by NOTCH3 mutations, NOTCH3 protein accumulation in arteries, and granular osmiophilic material. The mechanisms underlying CADASIL protein accumulation are currently unknown. We test the hypothesis that post-translational modification of NOTCH3 populations occurs within CADASIL cerebral vasculature. Methods: We generated monoclonal antibodies against the N-terminal region of NOTCH3. Antibodies were used for immunohistochemistry on 19 CADASIL and 6 age-matched control brains. Epitope mapping was performed on defined NOTCH3 fragments. Proteins treated with reducing agents were scored for antibody reactivity by western blotting. NOTCH3 complex formation was tested by co-immunoprecipitation. Results: NOTCH3 ectodomain undergoes cleavage into an N-terminal fragment (NTF) in diseased human cerebral vessels. The site of fragmentation was mapped to the peptide bond following D80. Mutagenesis of P81 abolished fragmentation. Both acidic and reducing conditions promote NOTCH3 cleavage in vitro . Finally, NTF is capable of forming dimers and detergent-resistant complexes with full length NOTCH3. Conclusion: Our studies demonstrate non-enzymatic fragmentation of NOTCH3 in CADASIL that could drive protein aggregation events that hallmark CADASIL. As in Alzheimer’s disease, protein fragmentation may contribute to neuropathology. Future studies will include testing the pathogenicity of fragmented NOTCH3, a potential novel target for cSVD therapy.

BACKGROUND Angioscopy - or endovascular endoscopy - is a catheter-based technique employing a flexible fiberoptic angioscope to directly visualize arterial lumen. Poor resolution and excessive stiffness of pre-existent angioscopes limited their use clinically. Recent advances resulted in novel fused optical fiber bundle angioscopes with improved flexibility and imaging resolution. Use of these devices in endovascular neurosurgery is still largely unexplored. OBJECTIVE To evaluate image quality and feasibility of optical angioscopes for diagnostic and interventional neuro-angioscopy in carotid arteries of human cadavers. METHODS A 5-F optical angioscope was used in human cadaveric carotid arteries to inspect integrity of arterial walls, identify atherosclerotic plaques and associated lesions prone to thrombogenicity, place intravascular occlusion coils, and deploy endovascular stents with real-time visualization. RESULTS Angioscopy provided key information about endoluminal anatomy such as presence and characteristics of atherosclerotic plaques and thrombogenic lesions not detected by conventional diagnostic methods. Direct real-time visualization of vascular lumen during endovascular interventions provided information on spatial distribution of coils, coil loop herniation, and apposition of stent cells against carotid artery wall complementary to angiography. CONCLUSIONS Fused optical fiber bundle angioscopes provide good-quality endoluminal images in human carotid arteries. Their use can feasibly assist in navigation of extracranial carotid arteries to inspect integrity of the arterial wall and identify atherosclerotic plaques and associated lesions vulnerable to thrombogenicity, allow placement of intravascular occlusion coils, and assess apposition of stents to vessel wall. Further in Vivo validation needs to be conducted along with additional research to improve image quality, flexibility, and size of angioscopes.

Cerebral small vessel disease is a common condition linked to dementia and stroke. As an age-dependent brain pathology, cerebral SVD may share molecular processes with core neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Many neurodegenerative diseases feature abnormal protein accumulation and aberrant protein folding, resulting in multimerization of specific proteins. We investigated if a small NOTCH3 N-terminal fragment (NTF) that co-registers with pathologically affected cells in the inherited SVD, CADASIL, is capable of multimerization. We also characterized endogenous small molecule vascular enhancers and inhibitors of multimerization. NTF multimerizes spontaneously and also forms conjugates with vascular catecholamines, including dopamine and norepinephrine, which avidly promote multimerization of the protein. Inhibition of catecholamine-dependent multimerization by vitamin C and reversal by reducing agents implicate an essential role of oxidation in NTF multimerization. Antibodies that react with degenerating arteries in CADASIL tissue preferentially bind to multimerized forms of NTF. These studies suggest that multimerization of proteins in the aging brain is not restricted to neuronal molecules and may participate in age-dependent vascular pathology.

Vascular smooth muscle cells (SMCs) undergo a series of dramatic changes in CADASIL, the most common inherited cause of vascular dementia and stroke. NOTCH3 protein accumulates and aggregates early in CADASIL, followed by loss of mature SMCs from the media of brain arteries and marked intimal proliferation. Similar intimal thickening is seen in peripheral arterial disease, which features pathological intimal cells including proliferative, dedifferentiated, smooth muscle-like cells deficient in SMC markers. Limited studies have been performed to investigate the differentiation state and location of SMCs in brain vascular disorders. Thus, we investigated the distribution of cells expressing SMC markers in a group of genetically characterized, North American CADASIL brains. We quantified brain RNA abundance of these markers in nine genetically verified cases of CADASIL and found that mRNA expression for several mature SMC markers was increased in CADASIL brain compared to age-matched control. Immunohistochemical studies and in situ hybridization localization of mRNA demonstrated loss of SMCs from the arterial media, and SMC marker-expressing cells were instead redistributed into the intima of diseased arteries and around balloon cells of the degenerating media. We conclude that, despite loss of medial smooth muscle cells in diseased arteries, smooth muscle markers are not lost from CADASIL brain, but rather, the localization of cells expressing mature SMC markers changes dramatically.

Cerebral small-vessel disease is a prevalent condition that is strongly associated with ischemic stroke and dementia. The most prevalent inherited cause of cerebral small-vessel disease is CADASIL, cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy, a disorder linked to mutations in NOTCH3. The most common symptoms of CADASIL are small ischemic strokes and/or transient ischemic attacks and cognitive impairment, appearing in middle age, that may progress to frank vascular dementia. However, it is increasingly recognized that individual symptom types, onset, and disease severity span a wide spectrum, even among individuals in the same family. Magnetic resonance imaging in CADASIL reveals severe white-matter hyperintensities, evidence of prior subcortical strokes, and, in some cases, microhemorrhages. Several hundred mutations in NOTCH3 have been described worldwide in CADASIL, and virtually all of these mutations alter the cysteine content of the extracellular NOTCH3 gene product. This molecular genetic signature of CADASIL has led to the hypothesis that structural abnormalities in the vascular smooth-muscle protein NOTCH3 trigger arterial degeneration, vascular protein accumulation, and cerebrovascular failure.

Regulation of cerebral endothelial cell function plays an essential role in changes in blood-brain barrier permeability. Proteins that are important for establishment of endothelial tight junctions have emerged as critical molecules, and PDZ domain containing-molecules are among the most important. We have discovered that the PDZ-domain containing protein periaxin (PRX) is expressed in human cerebral endothelial cells. Surprisingly, PRX protein is not detected in brain endothelium in other mammalian species, suggesting that it could confer human-specific vascular properties. In endothelial cells, PRX is predominantly localized to the nucleus and not tight junctions. Transcriptome analysis shows that PRX expression suppresses, by at least 50%, a panel of inflammatory markers, of which 70% are Type I interferon response genes; only four genes were significantly activated by PRX expression. When expressed in mouse endothelial cells, PRX strengthens barrier function, significantly increases transendothelial electrical resistance (~35%; p

The human cerebrovascular system is responsible for regulating demand-dependent perfusion and maintaining the blood-brain barrier (BBB). In addition, defects in the human cerebrovasculature lead to stroke, intracerebral hemorrhage, vascular malformations, and vascular cognitive impairment. The objective of this study was to discover new proteins of the human cerebrovascular system using expression data from the Human Protein Atlas, a large-scale project which allows public access to immunohistochemical analysis of human tissues. We screened 20,158 proteins in the HPA and identified 346 expression patterns correlating to blood vessels in human brain. Independent experiments showed that 51/52 of these distributions could be experimentally replicated across different brain samples. Some proteins (40%) demonstrated endothelial cell (EC)-enriched expression, while others were expressed primarily in vascular smooth muscle cells (VSMC; 18%); 39% of these proteins were expressed in both cell types. Most brain EC markers were tissue oligospecific; that is, they were expressed in endothelia in an average of 4.8 out of 9 organs examined. Although most markers expressed in endothelial cells of the brain were present in all cerebral capillaries, a significant number (21%) were expressed only in a fraction of brain capillaries within each brain sample. Among proteins found in cerebral VSMC, virtually all were also expressed in peripheral VSMC and in non-vascular smooth muscle cells (SMC). Only one was potentially brain specific: VHL (Von Hippel-Lindau tumor suppressor). HRC (histidine rich calcium binding protein) and VHL were restricted to VSMC and not found in non-vascular tissues such as uterus or gut. In conclusion, we define a set of brain vascular proteins that could be relevant to understanding the unique physiology and pathophysiology of the human cerebrovasculature. This set of proteins defines inter-organ molecular differences in the vasculature and confirms the broad heterogeneity of vascular cells within the brain.

The complex nature of atherosclerosis demands high-resolution approaches to identify subtle thrombogenic lesions and define the risk of plaque rupture. Here, we report the proof-of-concept use of a multimodal scanning fiber endoscope (SFE) consisting of a single optical fiber scanned by a piezoelectric drive that illuminates tissue with red, blue, and green laser beams, and digitally reconstructs images at 30 Hz with high resolution and large fields-of-view. By combining laser-induced reflectance and fluorescence emission of intrinsic fluorescent constituents in arterial tissues, the SFE allowed us to co-generate endoscopic videos with a label-free biochemical map to derive a morphological and spectral classifier capable of discriminating early, intermediate, advanced, and complicated atherosclerotic plaques. We demonstrate the capability of scanning fiber angioscopy for the molecular imaging of vulnerable atherosclerosis by targeting proteolytic activity with a fluorescent probe activated by matrix metalloproteinases. We also show that the SFE generates high-quality spectral images in vivo in an animal model with medium-sized arteries. Multimodal laser-based angioscopy could become a platform for the diagnosis, prognosis, and image-guided therapy of atherosclerosis.

Small penetrating brain artery thickening is a major feature of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Although affected fibrotic arteries of CADASIL have been shown to accumulate collagen, other components that compose pathological arterial walls remain incompletely characterized. We investigated the expression of decorin (DCN), the first collagen-binding small leucine-rich proteoglycan identified, in CADASIL. DCN was markedly upregulated in pathologically affected leptomeningeal and small penetrating arteries in CADASIL and was notably weaker in normal arteries from control brains. DCN protein was localized principally to the media and adventitia and only occasionally expressed in the intima. Immunoblotting of brain lysates showed a three-fold increase of DCN in CADASIL brains (compared with controls). Messenger RNA encoding DCN was five-fold increased in CADASIL. We conclude that DCN is the first identified proteoglycan to be identified in CADASIL arteries and may accumulate through transcriptional mechanisms. Additional studies are warranted to determine whether DCN localizes broadly to pathological small vessels in other cerebrovascular disorders.

In humans, ischemic stroke is associated with reduced heart rate variability (HRV), a predictor of neurogenic cardiac events. This study was conducted to determine whether experimental stroke preferentially affects HRV during specific times of the day or parts of the sleep-wake cycle. A continuous telemetry system was used to analyze electroencephalograms (EEG), electromyograms (EMG), and electrocardiograms (ECG) after experimental ischemic stroke in rats (n = 13). Animals were implanted with telemetry probes and, after a two week stabilization period, 72h of baseline continuous EEG, EMG, ECG data were collected. Animals were then assigned to two procedure groups: 1) experimental stroke via 90 minute intraluminal filament occlusion of the right middle cerebral artery, or 2) sham surgery. Continuous waveform recording and HRV analysis was then continued for 48h following each procedure. HRV analysis was conducted by both time-domain and frequency-domain methods. Heart rate exhibited circadian rhythms among all animals prior to and following both procedures. Neither procedure contributed to significant heart rate changes. We did not find sleep state or circadian-dependent alterations in HRV in baseline recording. However, after stroke, animals exhibited elevated high frequency (HF) HRV (>10% increase, p

Diverse preconditioning (PC) stimuli protect against a wide variety of neuronal insults in animal models, engendering enthusiasm that PC could be used to protect the brain clinically. Candidate clinical applications include cardiac and vascular surgery, after subarachnoid hemorrhage, and prior to conditions in which acute neuronal injury is anticipated. However, disappointments in clinical validation of multiple neuroprotectants suggest potential problems translating animal data into successful human therapies. Thus, despite strong promise of preclinical PC studies, caution should be maintained in translating these findings into clinical applications. The Stroke Therapy Academic Industry Roundtable (STAIR) working group and the National institute of Neurological Diseases and Stroke (NINDS) proposed working guidelines to improve the utility of preclinical studies that form the foundation of therapies for neurological disease. Here, we review the applicability of these consensus criteria to preconditioning studies and discuss additional considerations for PC studies. We propose that special attention should be paid to several areas, including 1) safety and dosage of PC treatments; 2) meticulously matching preclinical modeling to the human condition to be tested; and 3) timing of both the initiation and discontinuation of the PC stimulus relative to injury ictus.

Arteries in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) are susceptible to smooth muscle loss and fibrosis, but the molecular components underlying these dramatic vascular changes are not well characterized. The purpose of this study was to investigate the distribution of collagen isoforms in the cerebral vessels of North American CADASIL patients with classical NOTCH3 mutations. Expression of types I-VI collagen in brains obtained at autopsy from six CADASIL patients with cysteine-altering mutations in NOTCH3 was compared to control brain expression. We identified a consistent increase of types I, III, IV, and VI collagen in CADASIL brains. Strong accumulation of types I, III, IV and VI collagen was noted in all calibers of vessels, including small and medium-sized leptomeningeal arteries, small penetrating white matter arteries, and capillaries. Within leptomeningeal arteries, where we could define the three tunicae of each vessel, we found distinct collagen subtype distribution patterns in CADASIL. Types I and III collagen were largely found in either adventitial/medial or transmural locations. Type IV collagen was strictly intimal/medial. Type VI collagen was adventitial or adventitial/medial. Within the thickened penetrating arteries of CADASIL patients, all four collagens extended through most of the arterial wall. We observed increased staining of capillaries in CADASIL for types I, IV, and VI collagen. In conclusion, brain vascular collagen subtypes are increased in CADASIL in multiple layers of all sizes of arteries, with disease-specific changes most prominent in the tunica media and thickened small penetrating vessels. In diseased arteries, types I, III, and VI collagen spreads from an external location (adventitia) into the vascular media, while type IV collagen accumulates in an internal pattern (intima and media). These observations are consistent with a pathological role for collagen accumulation in the vascular media in CADASIL.

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit (NVU). We hypothesized that astrocytes and neurons generated from human induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance (TEER), reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein (PGP) efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of 1 neuron: 3 astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the NVU in both healthy and diseased humans.

Background and Purpose— Mineralocorticoid receptor (MR) antagonists have protective effects in rodent models of ischemic stroke, but the cell type-specific actions of these drugs are unknown. In the present study, we examined the contribution of myeloid cell MR during focal cerebral ischemia using myeloid-specific MR knockout mice. Methods— Myeloid-specific MR knockout mice were subjected to transient (90 minutes) middle cerebral artery occlusion followed by 24 hours reperfusion (n=5 to 7 per group). Ischemic cerebral infarcts were identified by hematoxylin and eosin staining and quantified with image analysis software. Immunohistochemical localization of microglia and macrophages was performed using Iba1 staining, and the expression of inflammatory markers was measured after 24 hours of reperfusion by quantitative reverse transcription–polymerase chain reaction. Results— Myeloid-specific MR knockout resulted in a 65% reduction in infarct volume ( P =0.005) after middle cerebral artery occlusion. This was accompanied by a significant reduction in activated microglia and macrophages in the ischemic core. Furthermore, myeloid-specific MR knockout suppressed classically activated M1 macrophage markers tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and interleukin-6 at the same time as partially preserving the induction of alternatively activated, M2, markers Arg1, and Ym1. Conclusions— These data demonstrate that myeloid MR activation exacerbates stroke and identify myeloid MR as a critical target for MR antagonists. Furthermore, these data indicate that MR activation has an important role in controlling immune cell function during the inflammatory response to stroke.

This paper analyzes the performance of a system consisting of a central base transmitter transmitting to a group of mobiles with on-off orthogonal modulation over a fast fading channel. Because the channel is changing across the symbol interval, inter-code interference appears at the output of each user's matched filter. The cumulative distribution function at one user's matched filter output is derived. From this distribution, a metric is introduced to evaluate which pairs of codes lead to the most severe inter-code interference for a particular fading spectrum.

Objective The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. Methods The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1 −/− ( NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1 −/− and wild type cells. Results The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1 −/− NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1 −/− NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1 −/− CVPCs, the expression of ISL1 , NKX2. 5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1 −/− ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. Conclusions NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve.