A Study That Collects Participant Data and Biospecimens to Analyze Pathogenic Exosomes That Mediate Increased Vascular Dementia Risk in Individuals With Herpes Zoster.

Vascular dementia: Vascular dementia is the second most common form of dementia after Alzheimer's disease and is characterized broadly as a loss of blood supply to the brain and compromised blood-brain barrier (BBB) ultimately leading to brain atrophy and cognitive impairment. Cerebrovascular insults such as stroke, myocardial infarctions, inflammation, and hyperactive platelets (thrombosis) are contributors and risk factors for the development of vascular dementia. Recently viruses have been identified as potential risk factors.

Varicella zoster virus (VZV) and vascular dysfunction: VZV is an exclusively human, double-stranded DNA alphaherpesvirus that produces varicella (chickenpox) then establishes latency within sensory and autonomic ganglionic neurons in \>95% of adults in the U.S. With aging and immunosuppression, VZV reactivates in at least 1 in 3 individuals to produce herpes zoster (HZ; shingles) and other neurological diseases with or without rash. Despite the availability of two HZ vaccines, more than 1 million Americans develop HZ annually. Lesser known to the general public is the frequency of VZV reactivation without rash (zoster sine herpete). Given that VZV reactivates from ganglionic neurons across the entire neuraxis, essentially all organs and tissues, including the vasculature, are prone to infection and disease without the characteristic and diagnostic HZ rash. In fact, it is predicted that for every 1 case of HZ with rash, 7 individuals will reactivate with VZV without corresponding rash. Furthermore, the group has shown that individuals can still shed active virus in their saliva up to 12 years post-HZ suggesting at least a subset of individuals may have ongoing replication despite no rash; in this study 67% of patients still had active virus in their saliva at least 3 months post-rash. Thus, the burden of VZV disease, particularly duration, is likely grossly underestimated.

HZ is recognized as a stroke risk factor; HZ increases the risk of stroke up to 1 year post-HZ, with risk higher when HZ occurs in the ophthalmic distribution or in individuals \<40 years of age. Specifically, data pooled from 9 studies in the U.S., Europe, and Asia show that the relative risk for stroke after HZ was 1.78 (95% CI 1.70-1.88) for the first month following HZ, dropping progressively to 1.20 (95% CI 1.14-1.26) after 1 year. Stroke risk increased by a larger margin during the first month after a HZ ophthalmicus episode: relative risk was 2.05 (95% CI 1.82-2.31); and the stroke risk remained elevated one year after the acute episode. A recent U.S. retrospective study of 23,339 HZ individuals and 46,378 controls, investigators found that during the 8 week aggregate period, HZ individuals were significantly more likely to suffer a stroke: adjusted incidence rate ratios for all patients and patients aged 18 to 49 years were 1.40 (95% CI, 0.93-2.11) and 8.12 (95% CI, 0.93-71.27), respectively (p \< 0.05); risk returned to baseline by 1 year. The increased stroke risk in individuals with HZ who are \< 50 years of age, compared to all HZ individuals, is remarkable because these individuals do not qualify for the HZ vaccine.

While the mechanisms underlying VZV and vascular disease are not fully elucidated, histological and immunohistochemical studies of VZV-infected arteries, analysis of cerebrospinal fluid (CSF) from VZV vasculopathy patients for cytokines and matrix metalloproteinases (MMPs), and in vitro studies provide some clues to the pathogenesis of disease (reviewed in Nagel and Bubak, 2018). Specifically, upon VZV reactivation, virus travels along nerve fibers that terminate in the outermost adventitial layer of the artery where virus-infected adventitial fibroblasts elicit a robust inflammatory response involving neutrophils early in disease, and T cells and macrophages throughout the entire course of disease. VZV-infected cells, neutrophils, and other infiltrating immune cells produce activated MMPs, which degrade the extracellular matrix potentially leading to weakening of the vessel wall, aneurysm formation, and rupture. Soluble factors secreted by inflammatory cells have also been described to further contribute to vascular smooth muscle death and accumulation of myofibroblasts in the thickened intima, potentially leading to arterial occlusion. However, the "soluble factors" have not been characterized until recently by the investigator group (described below).

Role of exosomes in disease states: Circulating exosomes have emerged as important "soluble factors" that can affect cells remote from their site of origin. Exosomes are small extracellular vesicles (\~40-200 nm in diameter) of endosomal origin that carry cargo (proteins, nucleic acids, etc.) from their cells of origin to adjacent or distal cells for communication during normal and pathological states, regulating biological processes and response to disease. For example, exosomes derived from platelets, megakaryocytes, and peripheral blood mononuclear cells (PBMCs) can transfer CXCR4 and CCR5 to cells that lack these receptors, thereby increasing the number of susceptible cells for HIV infection. In other studies, exosomes released from nasopharyngeal carcinoma cells positive for EBV contain the viral latent membrane protein 1 and other viral/cellular factors that manipulate the tumor microenvironment to enhance tumor progression and alleviate immune responses to tumor cells. Furthermore, exosomes (and other extracellular vesicles) derived from:

1. breast cancer cells induce platelet activation and aggregation, contributing to cancer-associated thrombosis; and 2. VZV-infected primary human brain vascular adventitial fibroblasts mediate endothelial cell dysfunction and transmission of virions. The investigators have recently published a pilot study showing pathogenic plasma exosomes from patients with acute HZ are prothrombotic and proinflammatory. Specific proteins that were significantly elevated in HZ exosomes and involved in the platelet activation, signaling, and aggregation pathways included thrombospondin 1 (THBS1) caveolae-associated protein 2 (CAVIN-2), coagulation factor V (F5),56 and coagulation factor XIII A1 chain (F13A1)57 (Fig. 1). Cardiovascular system disease proteins elevated in HZ exosomes included calmodulin 1 (CALM1) and transthyretin (TTR) (Fig. 1). Elevated calmodulin 1 may contribute to oxidative stress associated with neural cell death after hypoxic-ischemic brain injury and is a predictor of poor stroke outcomes. Although increased serum transthyretin has been associated with improved ischemic stroke outcomes,60 transthyretin can also oligomerize and induce oxidative stress, inflammation and cell toxicity. Indeed, during HZ, amyloid-promoting factors are present in plasma, 8 potentially causing aggregation of increased circulating transthyretin to the toxic oligomeric form in arteries reminiscent of cerebral amyloid angiopathy (CAA).

Link between vascular dementia and VZV: As described above, VZV has been shown to be a causative agent of the aforementioned risk factors (stroke, vascular inflammation, etc.). Furthermore, a recent epidemiological study from two independent databases (FinnGen and UKB) found VZV reactivation significantly increases the risk of developing vascular dementia in particular (2.33 and 6.22, respectively; hazard ratios). A barrier in the acceptance of VZV and viruses in general to the development of vascular dementia and Alzheimer's disease is the lack or inconsistent presence of virus antigen/nucleic acids in the diseased tissues at time of death. While this can be potentially explained by a "hit-and-run" phenomenon (virus infects tissues, causes irreversible damage but is subsequently cleared), the investigators propose that non-infectious exosomes produced in the periphery (dorsal root and trigeminal ganglia) can contribute to disease distant from site of viral replication. The mechanisms in which VZV directly contributes to vascular dementia pathology in the absence of a lytic infection has not been studied and is the focus of this proposal. Taken together, the investigators hypothesize that circulating exosomes during HZ and in subsequent months will promote vasculitis and thrombosis, thus providing a mechanistic basis for the increased vascular dysfunction risk preceding vascular dementia.

SIGNIFICANCE: Strong epidemiological and correlational data exists linking VZV and dementia, including vascular disease and Alzheimer's disease. However, a paucity of mechanistic evidence has resulted in a hesitancy to causatively link the two. Thus, this proposal aims to address this gap in knowledge by systematically testing the role of pathogenic, long-lasting exosomes following HZ on vascular disease processes. Mechanistically uncovering proteins/miRNAs of interest can potentially change clinical practice with regards to duration of antiviral therapy and use of anti-platelet agents for HZ, as well as increase support for vaccination to prevent rash and mitigate stroke and vascular dementia risk. Furthermore, findings will serve as a model for how other pathogens can have long-lasting effects at sites remote from regions of infection and stimulate further research on the biological consequences of pathogen-associated exosomes.

INNOVATION AND IMPACT: This proposal has several areas of innovation. The longitudinal characterization and mechanistic investigation of circulating exosomes during HZ and 12-months post will:

1. provide a mechanistic understanding to the epidemiologically observed increased risk of vascular dementia and stroke following HZ. The direct contributions of viruses to dementia, including vascular dementia and Alzheimer's disease, is still controversial given most data linking the two are epidemiological and correlational. Data generated in this proposal will provide unbiased mechanistic information on how VZV can directly lead to vascular disease, contributing much-needed causative evidence of an early-stage risk factor or lack thereof. 2. change clinical management. Currently, modifiable stroke and vascular disease risk factors include hypertension, smoking, diabetes and hypercholesterolemia. Because these pathogenic exosomes increase risk of thrombosis and vasculitis, stroke risk factor modification should include HZ vaccination with the non-live subunit vaccine (Shingrix). If HZ occurs, longer duration antiviral therapy may be warranted as determined by results showing the length of time these pathogenic exosomes persist, as well as anti-platelet and anti- inflammatory agents. When available, there may also be a use for novel therapeutics that prevent exosome release/fusion. 3. change the paradigm of infectious disease. Typically, the investigators assume that once the clinical features of an infectious agent have resolved (such as rash from VZV reactivation), its subsequent impact on human health is negligible. The presence of pathogenic exosomes circulating for months in the absence of the triggering infectious agent suggests that clinicians need to consider infectious history remote from the time of stroke or vascular disease presentation. 4. employ new, cutting edge technology (ExoView R200) that will allow sorting and characterization of single exosomes to help us understand the population heterogeneity. These results, along with the larger body of results from the 2 Aims (including exosome sequencing data), will provide data for future grants to elucidate the function and cells of origin of the exosome subsets.

Study Rationale Aim 1. Analyze exosome content for known vascular damaging proteins/miRNAs from matched control and HZ individuals over a 12-month period. Using mass spectrometry and small RNA sequencing, the investigators will quantify abundance and temporal changes of exosomal proteins and miRNAs associated with vascular dysfunction, inflammation, coagulation, and stroke, then correlate analytes with clinical and la

• Determine the mechanism(s) in which non-infectious HZ exosomes promote vascular dysfunction. [Срок оценки: From enrollment throughout the 5-year proposal period, occurring in a cyclical and overlapping manner.]