The vascular clock: a new insight into endothelial cells and pericytes crosstalk

Background/Introduction Circadian rhythms, defined as biological oscillations with a period of circa 24h, regulate many physiological processes in the cardiovascular system, such as vascular function, vascular tone, blood pressure, heart rate and thrombus formation [1]. The vasculature responds to the main pacemaker located in the brain, but it also possesses its own clock. Indeed, a molecular clock has been identified in endothelial cells (EC) and smooth muscle cells (SMC). The disruption of the circadian clock profoundly affects cardiovascular functionality with adverse cardiovascular events such as myocardial infarction or stroke showing a 24h rhythmicity with a peak incidence in the early morning. Among several mechanisms affected by circadian dysregulation, angiogenesis plays a fundamental role in homeostasis and development of new blood vessels. EC and pericytes (PC) are the two main cell populations in the capillaries, and their physical and paracrine interaction drives and regulates the sprouting. However, the presence and the role of circadian rhythms in pericytes and whether the molecular clock affects the endothelial/pericyte interactions remain unexplored. Purpose The aim of this study is to identify a molecular clock in human vascular pericytes and elucidate the impact of the circadian clock on the formation of new blood vessels. Methods Human primary PC were synchronised and the rhythmicity of clock genes measured by luminescence, immunofluorescence, and qPCR. Synchronised PC were co-cultured with Bmal1::LUC human primary EC. The effect of PC synchronisation and circadian clock disruption by shRNA on EC clock genes and angiogenic potential were measured by luminescence and Matrigel assay, respectively. A macroporous polyurethane scaffold was developed for 3D co-cultures. Results PC presented rhythmic expression of the principal circadian genes with a circa 24h period but in our experimental setting, EC did not show circadian rhythmicity. Synchronised PC supported the rhythmic expression of the clock gene Bmal1 in EC in a contact co-culture system, suggesting a secondary form of EC molecular clock regulation. Non-contact co-cultures failed to synchronise EC. Furthermore, when the clock was disrupted in PC, their capacity to support EC's tube-forming capacity on Matrigel was impaired; clock disruption in EC did not affect angiogenesis, supporting the hypothesis that a disrupted clock in perivascular cells affects angiogenesis. In a 3D tissue engineering scaffold seeded with both EC and PC, the synchronisation of the clock led to the development of organised vascular-like structures around the scaffold's pores, as compared to the non-synchronised condition where cells appeared disorganised. Conclusion This study defines for the first time the existence of an endogenous molecular circadian clock in perivascular cells and suggests implications for circadian clock synchronisation in physiological and therapeutic angiogenesis. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey Doctoral CollegeUniversity of Surrey Bioprocess and Biochemical Engineering (BioProChem) Group.

Telocytes and Lymphatics of the Human Colon

Background: Telocytes (TCs) are a peculiar morphological type of stromal cells. They project long and moniliform telopodes, visible on various bidimensional sections. Originally regarded as “interstitial Cajal-like cells”, gastrointestinal TCs were CD34+. Further double-labelling studies found that colon TCs are negative for the expressions of the PDGFR-α and α-SMA. However, the TCs in colon were not distinguished specifically from endothelial cells (ECs), vascular or lymphatic. A combinational approach is important for accurate TC identification. Hence, we designed an immunohistochemical study of human colon to check whether ECs and CD34+ TCs express different markers. Methods: Immunohistochemistry was performed on archived paraffin-embedded samples of human colon (nine cases) for the following markers: CD31, CD34, CD117/c-kit and D2-40 (podoplanin). Results: A distinctive population of CD34+ TCs was found coating the myenteric ganglia. However, also perivascular cells and vascular ECs were CD34+. c-kit expression was equally found in interstitial Cajal cells (ICCs) and perivascular cells. The CD34 TCs did not express c-kit. As they were equally CD31- and D2-40- they were assessed as different from ECs. Conclusions: Testing specific markers of ECs, vascular and lymphatic, in the same tissues in which CD34+ TCs are found, is much more relevant than to identify TCs by transmission electron microscopy alone.

Pericyte-derived fibrotic scarring is conserved across diverse central nervous system lesions

Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.

Blood-Brain Barrier Dysfunction Amplifies the Development of Neuroinflammation: Understanding of Cellular Events in Brain Microvascular Endothelial Cells for Prevention and Treatment of BBB Dysfunction

Neuroinflammation is involved in the onset or progression of various neurodegenerative diseases. Initiation of neuroinflammation is triggered by endogenous substances (damage-associated molecular patterns) and/or exogenous pathogens. Activation of glial cells (microglia and astrocytes) is widely recognized as a hallmark of neuroinflammation and triggers the release of proinflammatory cytokines, leading to neurotoxicity and neuronal dysfunction. Another feature associated with neuroinflammatory diseases is impairment of the blood-brain barrier (BBB). The BBB, which is composed of brain endothelial cells connected by tight junctions, maintains brain homeostasis and protects neurons. Impairment of this barrier allows trafficking of immune cells or plasma proteins into the brain parenchyma and subsequent inflammatory processes in the brain. Besides neurons, activated glial cells also affect BBB integrity. Therefore, BBB dysfunction can amplify neuroinflammation and act as a key process in the development of neuroinflammation. BBB integrity is determined by the integration of multiple signaling pathways within brain endothelial cells through intercellular communication between brain endothelial cells and brain perivascular cells (pericytes, astrocytes, microglia, and oligodendrocytes). For prevention of BBB disruption, both cellular components, such as signaling molecules in brain endothelial cells, and non-cellular components, such as inflammatory mediators released by perivascular cells, should be considered. Thus, understanding of intracellular signaling pathways that disrupt the BBB can provide novel treatments for neurological diseases associated with neuroinflammation. In this review, we discuss current knowledge regarding the underlying mechanisms involved in BBB impairment by inflammatory mediators released by perivascular cells.

The emerging role of perivascular cells (pericytes) in viral pathogenesis

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.

Tumour-associated Angiogenesis and Intermediate Blood Vessels in Renal Cell Carcinoma

Background/Aim: Renal cell carcinoma is strongly vascularized, and formation of new blood vessels is a complex and multi-step process. In this study, we analysed the subtypes of intermediate blood vessels, as shown by double immunohistochemistry. Materials and Methods: Tumour-associated blood vessels were identified by double immunostaining based on CD34 and smooth muscle cell actin. Blood vessels were classified both quantitatively and qualitatively based on the expression of the aforementioned two markers. The main criteria to sub-classify intermediate blood vessels was the presence, distribution, and arrangement of perivascular cells. Results: We described three subtypes of intermediate blood vessels found particularly in the tumour area: Subtype 1 lacked perivascular cells, subtype 2 showed scattered pericytes attached to the vascular wall, and subtype 3 showed a continuous layer of perivascular cells on one side. Conclusion: We describe for the first time three subtypes of renal cell carcinoma-associated intermediate blood vessels, which could be important in prognosis and as potential targets for anti-vascular therapy.

O-065 The naughty cells of the endometriumxx

Stem/progenitor cells are the naughty cells of the endometrium! The term “naughty” has a number of connotations, one being immaturity which I will apply to the rare stem/progenitor cell populations hiding in the endometrium, where they have eluded scientists for so long. Despite their rarity, these immature cells have the capability of growing up and differentiating into the functional cells of the endometrium, producing their progenies in the process. The self-willed human endometrial epithelial progenitor cells (eEPC) and mesenchymal stem cells (eMSC) first revealed themselves through their clonogenic activity, shunning their mates and setting up clones of cells on their own. Their risqué production of identical copies of themselves ensures their continuity, much to the chagrin of their mature counterparts. They are sneaky and can produce large numbers of mature progeny, but rarely proliferate themselves preferring to take life easy and do little. They also spit out viability dyes (Hoechst) at a greater rate than mature endometrial cells to become Side Population (SP) cells. A number of approaches have been used to tame these naughty endometrial stem/progenitors. In order to determine the identity and location of these elusive cells, specific markers had to be found. The immature endometrial epithelial progenitor cells play tricks with the specific markers they express. For example, clonogenic eEPC are N-cadherin+, an epithelial mesenchymal transition marker, found by unbiassed gene profiling, revealed their hiding place in the bases of glands deep in the endometrial basalis. Similarly, SSEA-1+ basalis epithelial progenitors pirated their marker from mature neutrophils and differentiating human pluripotent stem cells. In mice the stem/progenitor cells like to play chase, with lineage tracing of individual genetically marked cells revealing their location in the intersection zone of the glands and luminal epithelium, and also in the gland bases (Axin2+ and Lgr5+). The identity of eMSCs has also been determined by discovery of specific markers, but even here the eMSC play games in human endometrium where sometimes they are pericytes (CD140b and CD146 double positive cells), sometimes perivascular cells (SUSD2+) and sometimes CD34+ cells in the adventitia of blood vessels. They are also adventitial perivascular cells in ovine endometrium, but this time they are CD271+. Mature endometrial stromal cell progeny are also naughty, often pretending to be eMSC, particularly when shed into menstrual fluid, confusing many of their status. Adding further to their misbehaviour, they express the same official MSC surface markers. To get even immature endometrial MSC strike back, claiming immunomodulatory properties in attempt to upstage their mature stromal progeny, also endowed with these properties. Finally, other endometrial cells such as macrophages may also be naughty as their mischievousness in evading detection can trick us to consider them as stem cells from the bone marrow, masquerading as endometrial epithelial or stromal cells. Naughty implies behaving badly and I will show data suggesting that stem/progenitor cells may escape the endometrium to cause a nasty disease, endometriosis. They may also become wayward and unruly, invading the myometrium to form adenomyosis. Some naughty epithelial progenitors defiantly pick up mutations to become cancer stem cells and initiate endometrial cancer. They may also malfunction because they do not obey estrogen signalling instructions, failing to proliferate and causing thin unresponsive endometrium. In their naughtiness, they may run away or get totally lost, thereby resulting in Asherman’s syndrome. Therefore, for numerous reasons, stem/progenitor cells are the naughty cells of the endometrium. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected].

Single-cell RNA sequencing of cultured human endometrial CD140b+CD146+ perivascular cells highlights the importance of in vivo microenvironment

Background Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear. Methods In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level. Results Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells. Conclusion In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.