Optical coherence tomography angiography (OCTA) performs non-invasive visualization and characterization of microvasculature in research and clinical applications mainly in ophthalmology and dermatology. A wide variety of instruments, imaging protocols, processing methods and metrics have been used to describe the microvasculature, such that comparing different study outcomes is currently not feasible. With the goal of contributing to standardization of OCTA data analysis, we report a user-friendly, open-source toolbox, OCTAVA (OCTA Vascular Analyzer), to automate the pre-processing, segmentation, and quantitative analysis of en face OCTA maximum intensity projection images in a standardized workflow. We present each analysis step, including optimization of filtering and choice of segmentation algorithm, and definition of metrics. We perform quantitative analysis of OCTA images from different commercial and non-commercial instruments and samples and show OCTAVA can accurately and reproducibly determine metrics for characterization of microvasculature. Wide adoption could enable studies and aggregation of data on a scale sufficient to develop reliable microvascular biomarkers for early detection, and to guide treatment, of microvascular disease.

Viruses may exploit the cardiovascular system to facilitate transmission or within-host dissemination, and the symptoms of many viral diseases stem at least in part from a loss of vascular integrity. The microvascular architecture is comprised of an endothelial cell barrier ensheathed by perivascular cells (pericytes). Pericytes are antigen-presenting cells (APCs) and play crucial roles in angiogenesis and the maintenance of microvascular integrity through complex reciprocal contact-mediated and paracrine crosstalk with endothelial cells. We here review the emerging ways that viruses interact with pericytes and pay consideration to how these interactions influence microvascular function and viral pathogenesis. Major outcomes of virus-pericyte interactions include vascular leakage or haemorrhage, organ tropism facilitated by barrier disruption, including viral penetration of the blood-brain barrier and placenta, as well as inflammatory, neurological, cognitive and developmental sequelae. The underlying pathogenic mechanisms may include direct infection of pericytes, pericyte modulation by secreted viral gene products and/or the dysregulation of paracrine signalling from or to pericytes. Viruses we cover include the herpesvirus human cytomegalovirus (HCMV, Human betaherpesvirus 5), the retrovirus human immunodeficiency virus (HIV; causative agent of acquired immunodeficiency syndrome, AIDS, and HIV-associated neurocognitive disorder, HAND), the flaviviruses dengue virus (DENV), Japanese encephalitis virus (JEV) and Zika virus (ZIKV), and the coronavirus severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2; causative agent of coronavirus disease 2019, COVID-19). We touch on promising pericyte-focussed therapies for treating the diseases caused by these important human pathogens, many of which are emerging viruses or are causing new or long-standing global pandemics.

Dengue is the most prevalent arthropod-borne viral disease affecting humans, with severe dengue typified by potentially fatal microvascular leakage and hypovolemic shock. Blood vessels of the microvasculature are composed of a tubular structure of endothelial cells ensheathed by perivascular cells (pericytes). Pericytes support endothelial cell barrier formation and maintenance through paracrine and contact-mediated signaling and are critical to microvascular integrity. Pericyte dysfunction has been linked to vascular leakage in noncommunicable pathologies such as diabetic retinopathy but has never been linked to infection-related vascular leakage. Dengue vascular leakage has been shown to result in part from the direct action of the secreted dengue virus (DENV) nonstructural protein NS1 on endothelial cells. Using primary human vascular cells, we show here that NS1 also causes pericyte dysfunction and that NS1-induced endothelial hyperpermeability is more pronounced in the presence of pericytes. Notably, NS1 specifically disrupted the ability of pericytes to support endothelial cell function in a three-dimensional (3D) microvascular assay, with no effect on pericyte viability or physiology. These effects are mediated at least in part through contact-independent paracrine signals involved in endothelial barrier maintenance by pericytes. We therefore identify a role for pericytes in amplifying NS1-induced microvascular hyperpermeability in severe dengue and thus show that pericytes can play a critical role in the etiology of an infectious vascular leakage syndrome. These findings open new avenues of research for the development of drugs and diagnostic assays for combating infection-induced vascular leakage, such as severe dengue. IMPORTANCE The World Health Organization considers dengue one of the top 10 global public health problems. There is no specific antiviral therapy to treat dengue virus and no way of predicting which patients will develop potentially fatal severe dengue, typified by vascular leakage and circulatory shock. We show here that perivascular cells (pericytes) amplify the vascular leakage-inducing effects of the dengue viral protein NS1 through contact-independent signaling to endothelial cells. While pericytes are known to contribute to noncommunicable vascular leakage, this is the first time these cells have been implicated in the vascular effects of an infectious disease. Our findings could pave the way for new therapies and diagnostics to combat dengue and potentially other infectious vascular leakage syndromes.

23Dengue is the most prevalent arthropod-borne viral disease affecting humans, with 24 severe dengue typified by potentially fatal microvascular leakage and hypovolaemic shock. 25Blood vessels of the microvasculature are composed of a tubular structure of endothelial cells 26 ensheathed by perivascular cells (pericytes). Pericytes support endothelial cell barrier 27 formation and maintenance through paracrine and contact-mediated signalling, and are critical 28 to microvascular integrity. Pericyte dysfunction has been linked to vascular leakage in 29 noncommunicable pathologies such as diabetic retinopathy, but has never been linked to 30 infection-related vascular leakage. Dengue vascular leakage has been shown to result in part 31 from the direct action of the secreted dengue virus (DENV) non-structural protein NS1 on 32 endothelial cells. Using primary human vascular cells, we show here that NS1 also causes 33 pericyte dysfunction, and that NS1-induced endothelial hyperpermeability is more pronounced 34 in the presence of pericytes. Notably, NS1 specifically disrupted the ability of pericytes to 35 support endothelial cell function in a 3D microvascular assay, with no effect on pericyte viability 36 or physiology. These effects are mediated at least in part through contact-independent 37 paracrine signals involved in endothelial barrier maintenance by pericytes. We therefore 38 identify a role for pericytes in amplifying NS1-induced microvascular hyperpermeability in 39 severe dengue, and thus show that pericytes can play a critical role in the aetiology of an 40 infectious vascular leakage syndrome. These findings open new avenues of research for the 41 development of drugs and diagnostic assays for combating infection-induced vascular 42 leakage, such as severe dengue.43 44 45 46 47 3 SIGNIFICANCE STATEMENT 48 The World Health Organisation considers dengue one of the top ten global public 49 health problems. There is no specific antiviral therapy to treat dengue virus and no way of 50 predicting which patients will develop potentially fatal severe dengue, typified by vascular 51 leakage and circulatory shock. We show here that perivascular cells (pericytes) amplify the 52 vascular leakage-inducing effects of the dengue viral protein NS1 through contact-53 independent signalling to endothelial cells. While pericytes are known to contribute to 54 noncommunicable vascular leakage, this is the first time these cells have been implicated in 55 the vascular effects of an infectious disease. Our findings could pave the way for new 56 therapies and diagnostics to combat dengue, and potentially other infectious vascular leakage 57 syndromes. 58 59 60 61 62 63 64 65 66 67 68 69 355 MR/R010315/1 to KM and a