Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Emerging molecular mechanisms of vascular dementia.

PURPOSE OF REVIEW: Microvascular ischemic disease of the brain is a common cause of cognitive impairment and dementia, particularly in the context of preexisting cardiovascular risk factors and aging. This review summarizes our current understanding of the emerging molecular themes that underlie progressive and irreparable vascular disease leading to neuronal tissue injury and dementia. RECENT FINDINGS: Cardiometabolic risk factors including diabetes and hypertension are known to contribute to vascular disease. Currently, the impact of these risk factors on the integrity and function of the brain vasculature has been target of intense investigation. Molecularly, the consequences associated with these risk factors indicate that reactive oxygen species are strong contributors to cerebrovascular dysfunction and injury. In addition, genetic linkage analyses have identified penetrant monogenic causes of vascular dementia. Finally, recent reports begun to uncover a large number of polymorphisms associated with a higher risk for cerebrovascular disease. SUMMARY: A comprehensive picture of key risk factors and genetic predispositions that contribute to brain microvascular disease and result in vascular dementia is starting to emerge. Understanding their relationships and cross-interactions will significantly aid in the development of preventive and intervention strategies for this devastating condition.

A multi-step transcriptional cascade underlies vascular regeneration in vivo.

The molecular mechanisms underlying vascular regeneration and repair are largely unknown. To gain insight into this process, we developed a method of intima denudation, characterized the progression of endothelial healing, and performed transcriptome analysis over time. Next-generation RNA sequencing (RNAseq) provided a quantitative and unbiased gene expression profile during in vivo regeneration following denudation injury. Our data indicate that shortly after injury, cells immediately adjacent to the wound mount a robust and rapid response with upregulation of genes like Jun, Fos, Myc, as well as cell adhesion genes. This was quickly followed by a wave of proliferative genes. After completion of endothelial healing a vigorous array of extracellular matrix transcripts were upregulated. Gene ontology enrichment and protein network analysis were used to identify transcriptional profiles over time. Further data mining revealed four distinct stages of regeneration: shock, proliferation, acclimation, and maturation. The transcriptional signature of those stages provides insight into the regenerative machinery responsible for arterial repair under normal physiologic conditions.

A personalized, multiomics approach identifies genes involved in cardiac hypertrophy and heart failure.

A traditional approach to investigate the genetic basis of complex diseases is to identify genes with a global change in expression between diseased and healthy individuals. However, population heterogeneity may undermine the effort to uncover genes with significant but individual contribution to the spectrum of disease phenotypes within a population. Here we investigate individual changes of gene expression when inducing hypertrophy and heart failure in 100 + strains of genetically distinct mice from the Hybrid Mouse Diversity Panel (HMDP). We find that genes whose expression fold-change correlates in a statistically significant way with the severity of the disease are either up or down-regulated across strains, and therefore missed by a traditional population-wide analysis of differential gene expression. Furthermore, those "fold-change" genes are enriched in human cardiac disease genes and form a dense co-regulated module strongly interacting with the cardiac hypertrophic signaling network in the human interactome. We validate our approach by showing that the knockdown of Hes1, predicted as a strong candidate, induces a dramatic reduction of hypertrophy by 80-90% in neonatal rat ventricular myocytes. Our results demonstrate that individualized approaches are crucial to identify genes underlying complex diseases as well as to develop personalized therapies.

NOTCH1 is a mechanosensor in adult arteries.

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell-cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.

A systems genetics approach identifies Trp53inp2 as a link between cardiomyocyte glucose utilization and hypertrophic response.

Cardiac failure has been widely associated with an increase in glucose utilization. The aim of our study was to identify factors that mechanistically bridge this link between hyperglycemia and heart failure. Here, we screened the Hybrid Mouse Diversity Panel (HMDP) for substrate-specific cardiomyocyte candidates based on heart transcriptional profile and circulating nutrients. Next, we utilized an in vitro model of rat cardiomyocytes to demonstrate that the gene expression changes were in direct response to substrate abundance. After overlaying candidates of interest with a separate HMDP study evaluating isoproterenol-induced heart failure, we chose to focus on the gene Trp53inp2 as a cardiomyocyte glucose utilization-specific factor. Trp53inp2 gene knockdown in rat cardiomyocytes reduced expression and protein abundance of key glycolytic enzymes. This resulted in reduction of both glucose uptake and glycogen content in cardiomyocytes stimulated with isoproterenol. Furthermore, this reduction effectively blunted the capacity of glucose and isoprotereonol to synergistically induce hypertrophic gene expression and cell size expansion. We conclude that Trp53inp2 serves as regulator of cardiomyocyte glycolytic activity and can consequently regulate hypertrophic response in the context of elevated glucose content.NEW & NOTEWORTHY Here, we apply a novel method for screening transcripts based on a substrate-specific expression pattern to identify Trp53inp2 as an induced cardiomyocyte glucose utilization factor. We further show that reducing expression of the gene could effectively blunt hypertrophic response in the context of elevated glucose content.

Systems Genetics Approach Identifies Gene Pathways and Adamts2 as Drivers of Isoproterenol-Induced Cardiac Hypertrophy and Cardiomyopathy in Mice.

We previously reported a genetic analysis of heart failure traits in a population of inbred mouse strains treated with isoproterenol to mimic catecholamine-driven cardiac hypertrophy. Here, we apply a co-expression network algorithm, wMICA, to perform a systems-level analysis of left ventricular transcriptomes from these mice. We describe the features of the overall network but focus on a module identified in treated hearts that is strongly related to cardiac hypertrophy and pathological remodeling. Using the causal modeling algorithm NEO, we identified the gene Adamts2 as a putative regulator of this module and validated the predictive value of NEO using small interfering RNA-mediated knockdown in neonatal rat ventricular myocytes. Adamts2 silencing regulated the expression of the genes residing within the module and impaired isoproterenol-induced cellular hypertrophy. Our results provide a view of higher order interactions in heart failure with potential for diagnostic and therapeutic insights.

Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model.

We aimed to understand the genetic control of cardiac remodeling using an isoproterenol-induced heart failure model in mice, which allowed control of confounding factors in an experimental setting. We characterized the changes in cardiac structure and function in response to chronic isoproterenol infusion using echocardiography in a panel of 104 inbred mouse strains. We showed that cardiac structure and function, whether under normal or stress conditions, has a strong genetic component, with heritability estimates of left ventricular mass between 61% and 81%. Association analyses of cardiac remodeling traits, corrected for population structure, body size and heart rate, revealed 17 genome-wide significant loci, including several loci containing previously implicated genes. Cardiac tissue gene expression profiling, expression quantitative trait loci, expression-phenotype correlation, and coding sequence variation analyses were performed to prioritize candidate genes and to generate hypotheses for downstream mechanistic studies. Using this approach, we have validated a novel gene, Myh14, as a negative regulator of ISO-induced left ventricular mass hypertrophy in an in vivo mouse model and demonstrated the up-regulation of immediate early gene Myc, fetal gene Nppb, and fibrosis gene Lgals3 in ISO-treated Myh14 deficient hearts compared to controls.

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1ß induced miRs-21-3p and -27a-5p in human aortic endothelial cells.

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1ß (IL-1ß). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1ß treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3' untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.

Mapping genetic contributions to cardiac pathology induced by Beta-adrenergic stimulation in mice.

BACKGROUND: Chronic stress-induced cardiac pathology exhibits both a wide range in severity and a high degree of heterogeneity in clinical manifestation in human patients. This variability is contributed to by complex genetic and environmental etiologies within the human population. Genetic approaches to elucidate the genetics underlying the acquired forms of cardiomyopathies, including genome-wide association studies, have been largely unsuccessful, resulting in limited knowledge as to the contribution of genetic variations for this important disease. METHODS AND RESULTS: Using the ß-adrenergic agonist isoproterenol as a specific pathological stressor to circumvent the problem of etiologic heterogeneity, we performed a genome-wide association study for genes influencing cardiac hypertrophy and fibrosis in a large panel of inbred mice. Our analyses revealed 7 significant loci and 17 suggestive loci, containing an average of 14 genes, affecting cardiac hypertrophy, fibrosis, and surrogate traits relevant to heart failure. Several loci contained candidate genes which are known to contribute to Mendelian cardiomyopathies in humans or have established roles in cardiac pathology based on molecular or genetic studies in mouse models. In particular, we identify Abcc6 as a novel gene underlying a fibrosis locus by validating that an allele with a splice mutation of Abcc6 dramatically and rapidly promotes isoproterenol-induced cardiac fibrosis. CONCLUSIONS: Genetic variants significantly contribute to the phenotypic heterogeneity of stress-induced cardiomyopathy. Systems genetics is an effective approach to identify genes and pathways underlying the specific pathological features of cardiomyopathies. Abcc6 is a previously unrecognized player in the development of stress-induced cardiac fibrosis.