Occurrence, (Micro)vascular dysfunction

The description of this KE aims to facilitate incorporation of evidence for distinct but relevant molecular level events influencing  individual molecules and cells, but also important interactions driving higher (tissue/organ) organisational level events. 

The complexity of the current KE description is driven by: the level of biological organisation that it aims to cover, and the increasing availability and complexity of the measurement methods that aim to address it, also reflected in the evidence available for the converging KER2831. The challenges with complex KEs and their representations have been discussed (10.14573/altex.2112161, 10.1093/toxres/tfaa099).  Re-organisation into sub-KE may be possible as the data organisation and visualisation methods are enhanced in the AOP Wiki and also as evidence form new assays grows, providing greater resolution of the mechanistic aspects of microvascular dysfunction.

Microvasculature (MV) is the tissue system/organ of vessels (capillaries, arterioles and venules), which enable delivery and exchange of gases (O2, CO2), nutrients, metabolites and circulating immune cells, within all organs of the body. Thus proper function of the MV is essential for adequate response to changes in metabolic demand and blood flow to the organs.

Functional response of the microvasculature to normal physiologically changing or stressed tissue/organ environment is mediated by its specific cellular and molecular structure consisting of a variety of cell types intimately linked to the tissue environment.

Exsosome-mediated communication between different cell types within the vasculature is increasingly recognised as key aspect of (mycro)vascular function, while interference/perturbations of this intercellular communication emerges as an important factor driving dysfunction and potential target for therapy [10.1186/s12964-022-00949-6; 10.1007/s12012-021-09700-y; 10.3389/fcvm.2022.912358; 10.3389/fcell.2019.00353; 10.1016/j.bbadis.2020.165833].

Depending on the tissue/organ type there may be subtle molecular differences but the general cellular outline of the MV can be illustrated as in Figure 1 include: 

Endothelium:  inner lining monolayer of closely juxtaposed squamous endothelial cells (ECs). The quiescent or non-proliferating endothelium has an active role in maintaining vascular homeostasis by receiving and generating diverse biochemical (autocrine, paracrine, and endocrine) and mechanical signals (Ricard et al., 2021-10.1038/s41569-021-00517-4).

One of the most commonly used surface markers for identification/sorting/enriching viable quiescent ECs is the CD31 in combination with the absence and/or presence of surface markers specific for other cells or for specific activation, dysfunction or differentiation state of the ECs (Goncharov 2017 - 10.1155/2017/9759735 Rakocevic 2017 - . 10.1016/j.yexmp.2017.02.005). A convenient list of specific endothelial cell markers and reagents for their identification can be found here together with some basic background information on each marker.

Dysregulated communication between ECs and other vascular cell types is associated with vascular dysfunction and pathological vascular remodelling in various pathological conditions (Rajendran et al., 2013 - 10.7150/ijbs.7502; Méndez-Barbero et al., 2021- 10.3390/ijms22147284). Rajendran et al 2013 - 10.7150/ijbs.7502, provides a good comparison of healthy and dysfunctional vasculature based on the biochemical products of (mainly) ECs including: nitric oxide (NO), prostacyclin (PGI2), reactive oxygen species (ROS), uric acid, plasminogen activator inhibitor 1 (PAI-1), von Willebrand factor (vWF), P-selectin. soluble vascular cell adhesion molecule (sVCAM.), soluble intercellular adhesion molecule (sICAM), E-selectin, C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6). Under some stress conditions (e.g. infections, cell aging and damage) vascular dysfunction can be triggered by specific interaction of the vascular components and the innate immune surveillance complement system (10.1038/nrneph.2016.70; 10.1111/cei.12952; Immunobiology: The Immune System in Health and Disease. 5th edition; 10.2340/00015555761316). Dysfunction triggered by complement activation is particularly relevant for infectious stressors initiating adoptive immunity as part of its normal host response (e.g. viruses) [10.1016/j.virol.2010.12.045; 10.3389/fimmu.2020.01450].

Given the dynamic responses of ECs to environmental signals, including these from the intimately connected perivascular cells, test systems based on ECs require careful phenotypic characterisation.

ECs structure and function exhibit significant tissue specificity. Single­cell RNA­seq atlas of mouse endothelial cells isolated from different tissues by flow cytometry without the cell culture step, identified transcriptomic signatures of quiescent ECs and found that: arterial and venous endothelial cells from a specific tissue clustered together, showing that vascular endothelial cell heterogeneity comes mainly from tissue specificity rather than arterial, capillary or venous identity (Kalucka 2020 - 10.1016/j.cell.2020.01.015). Moreover, capillary endothelial cells that are involved in gas, ion, metabolite and hormone exchange between the blood and tissues have the highest heterogeneity among tissues (Kalucka 2020 - 10.1016/j.cell.2020.01.015). Similar results (i.e. tissue specificity) have been reported after re-analysis of independently generated single cells sequencing data Paik 2020 - 10.1161/CIRCULATIONAHA.119.041433)

Comprehensive vasculature atlas from human tissues is not available but re-analysis of scRNAseq data from control human lung cohorts has been performed (Schupp 2021 - 10.1161/CIRCULATIONAHA.120.052318). The analysis identified that broad EC categories and conserved marker genes similar to those identified the mice data (Schupp 2021 - 10.1161/CIRCULATIONAHA.120.052318; Kalucka 2020 - 10.1016/j.cell.2020.01.015).

Intercellular communication of the endothelial with the other cells of the MV appears to be an important determinant of MV (dys)function (10.1016/j.bbadis.2020.165833; 10.1007/s12012-021-09700-y; 10.1038/ncomms9024; 10.1038/s41598-018-34357-z; 10.1021/acs.molpharmaceut.8b00765). EC derived exosomes contain some unique endothelial markers, including VE-cadherin, E-selectin, P-selectin, PECAM, ICAM-1, MCAM, endoglin, and ACE.  In addition, they also contain various miRNAs [10.1016/j.bbadis.2020.165833; 10.3389/fmolb.2020.619697].. All of these molecules have biological functions in both normal endothelial physiology and pathogenesis.

Glicocalix, on the lumen side the MV, generated by the endothelium is composed of a negatively charged network of GAGs and proteoglycans. It modulates interactions between the vasuclature wall and blood cell. Glicocalix  represents a binding site for crucial anticoagulant mediators such as heparin cofactor II, antithrombin III, thrombomodulin and tissue factor pathway inhibitor (TFPI). [Yilmaz 2019 - 10.1093/ckj/sfz042],

Basement membrane represents the layer of complex extracellular matrix (ECM) proteins (20–200 nm) on the tissue side the endothelium. It provides a mechanical support and divide tissues into compartments, but also influence cellular behaviour Vascular basement membrane is a three-dimensional network of proteins from four major glycoprotein families: laminins, collagen IV isoforms, nidogens, and heparan sulfate proteoglycans (HSPG). Additionally, many other proteins are differentially expressed in the vascular basement membrane depending on the developmental and physiological state of the vasculature. These include insoluble fibronectin, fibulin 1 and 2, collagen type XVIII, thrombospondins 1, and SPARC (secreted protein acidic and rich in cysteine) (Thomsen 2017 - 10.1177/0271678X17722436, and references therein). The ECM is generated by ECs and pericytes (Thomsen 2017 - 10.1177/0271678X17722436, and references therein).

Pericytes are perivascular, mural cells that have intimate contact with the endothelial cells and together support important functions such as maintaining the physical and functional integrity of the Blood Brain Barer (BBB), regulating capillary diameter, cerebral blood flow and maintaining extracellular matrix protein levels. Their identity, ontogeny, and progeny is not characterised as well as that of endothelial cells. They express multiple markers and their origin differs by tissue, which makes their identification and understanding of their function difficult (Armulik 2011 - 10.1016/j.devcel.2011.07.001).

A recent scRNAsec analysis of microfluidic droplets of mouse tissues confirm that two previously known perycite and conserved markers (Cspg4 or Pdgfrb) are co-expressed in the mural cell clusterdefined as perycites from lung, heart, kidney, and bladder (Beek 2022 -10.3389/fcvm.2022.876591). Other potential tissue specific markers were also identified in this study.

Pericytes also have the potential to give rise to different tissues in vitro but this is not clear in vivo. (Yamazaki 2018 - 10.3389/fcvm.2018.00078).

Vascular smooth muscle cells  [VSMC] surround the endothelium, pericytes and basal membrane in larger vessels. They contain contractile filaments and maintain vascular tone in response to (endocrine?, paracrine? autocrine?) action of vasoactive mediators and neurotransmitters (e.g. Angiotenisn II, Angiotensin 1-7, Endothelins, NO, epinephrine and norepinephrine) via their receptors or effectors e.g AGTR1, MasR, endothein receptor A and B or guanylate cyclase, and adrenoceptors, respectively.

Under different stress condition (persistant stretch, injury, inflammatory cytokines and excess oxidized lipids) and also during normal development, VSMC can undergo phenotypic switching or remodelling from a contractile to synthetic or proliferative phenotype wich involves a partial down regulation of the proteins that activate the contractile apparatus in favour of the synthetic and proliferative cellular machinery [10.5772/intechopen.77115; 10.1152/physrev.00041.2003].

Proliferative smooth muscle cells have an attenuated response to vasoconstrictors and vasodilators, probably due to the down regulation of the contractile apparatus and certain elements of the subcellular signalling machinery that is involved in vasoconstriction. Notably, many of the vasoactive modulators (e.g. angiotensin II endothelin and noradrenaline) also function to promote smooth muscle proliferation. Chronically elevated levels platelet derived growth factor (PDGF), for example, generated from unstable thrombus, can also contribute to proliferative vascular disorders. On the other hand, nitric oxide, limits smooth muscle hyperplasia and hypertrophy. ACE2, Angotenisn 1-7 and Mas receptor also appear to play important role in the modulation of the prliferative phenotypic switching of VSMC [10.1155/2012/121740; 10.1161/HYPERTENSIONAHA.114.03388; 10.1042/BSR20192012; 10.26355/eurrev_202004_20867].

Similar to the endothelial cells, VSMCs produce exosomes containing components of the Extracellular Matrix (ECM) such as collagens, proteoglycans, hyaluronan and laminin as well as matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMPs) which are particularly important for repair and remodelling of growing or damaged vessels. miRNAs released from VSMC also , are increasingly recognised as intercellular signalling molecules important for MV (dys)function [10.1186/s12964-022-00949-6; 10.1016/j.ymthe.2017.03.031]

Progenitor cells - [to complete]

Fig. 1: Cellular structure of small vasculature from Jin et al. 2020

MV is a dynamic organ and proper cellular differentiation, renewal and intercellular interactions mediated by various signalling molecules govern its functional and structural integrity. These interactions also govern the return to homeostasis under some stress conditions, restoring basal structure/function of the vasculature and ultimately well oxygenated tissue/organ.

In addition to the markers for specific MV cell lineage, other more ubiquitously expressed proteins (such as angiotensin converting enzyme 2 – ACE2) may, under some circumstances, represent markers for differentiation state or (dys)function of the MV at cellular or tissue level. For example, ACE2 expression in HBMEC & HUVEC perfusion culture is stimulated by flow (HBMEC < HUVEC) (qRT-PCR); also it is increased by flow intensity and vessel shape in the MCA 3D model of stenosis (immunostaining cells) [10.1161/STROKEAHA.120.032764]. Given that ACE2 is implicated as the main receptor for viral entry of SARS-CoV2 associated with COVID19 disease, the level and the dynamics of ACE2 expression is likely to be important for driving COVID19-associated vascular dysfunctions. Notably, in mature human tissues, ACE2 is not expressed at significant levels in the vascular compared to other cells evaluated (Human Protein Atlas version 22.0). Within the human vascular tissue it appears that expression in smooth muscle cells is significantly higher than that in endothelial cells and comparable to that in fibroblasts (Human Protein Atlas version 22 – single cell type-vascular). Pericytes are not specifically identified in this project.

Excessive disruption of the structural integrity of the MV or interference with the normal balanced function of molecular mediators leads to inability of the MV to maintain homeostasis i.e. (micro)vascular  dysfunction.

MV dysfunction can be evaluated and/or quantified by:

• Macroscopic and microscopic observations of the structural integrity of the structure of the MV (basic histochemical staining or immunostaining for specific cellular markers) [e.g. PMID 17974127; 10.1101/2020.08.19.251249; 10.1016/S2352-3026(20)30216-7, 10.1007/s00428-020-02886-6; 10.1038/s41379-021-00793-y; 10.1161/CIRCRESAHA.120.317447; 10.3390/diagnostics10080575; 10.1002/path.5549]

• Evaluation of the functional integrity of the barrier: Electric Cell-Substrate Impedance Sensing (ECIS), trans-endothelial electrical resistance (TEER), FITC-dextran permeability assays [e.g. [10.1016/j.nbd.2020.105131; 10.1152/ajplung.00223.2021; 10.3389/fcvm.2021.687783]

• Differential expression of surface adhesion molecules (eg. ICAM-1, VCAM-1) by FACS-SCAN assay [e.g. 10.1186/s13054-021-03631-4] or Western blotting [e.g. 110.3389/fcvm.2021.687783]

• Differential expression of pro-inflammatory and or anti-inflammatory cytokines by various RNA measurement assays, various immune based assays using specific antibodies) [10.1016/j.nbd.2020.105131; 10.1161/STROKEAHA.120.032764; 10.1172/JCI148635], including proteomic approaches [10.1172/JCI148635]

• Differential expression of matrix metalloproteinases (e.g. MMP2, MMP3, MMP9, MMP12) by various RNA measurement assays or various immune based assays using specific antibodies or fluorescent tags) [e.g [10.1016/j.nbd.2020.105131]

• Differential expression of coagulation and/or fibrinolytic factors (e.g. plasminogen activator inhibitor 1(PAI-1), plasminogen activator (tPA), urokinase (uPA)) by various RNA measurement assays, various immune based assays using specific antibodies, or various assays for their specific enzymatic activity. [e.g. [10.1165/rcmb.2020-0544OC] including proteomic approaches [10.1172/JCI148635]

• Detection of tissue/cell stress markers: (e.g. reactive oxygen species (ROS); prostaglandins (PG); vasoactive peptides, such as angiotensin II (Ang II), angiotensin (1-7) (Ang 1-7) or activity of their receptors 

• Detection of contractile factors, including endothelin (ET), thromboxane A2 (TXA2)

• Analysis and quantification of exosomal markers