Sex differences in the expression of cell adhesion molecules on microvesicles derived from cultured human brain microvascular endothelial cells treated with inflammatory and thrombotic stimuli

Abstract

Background There are sex differences in risk for stroke and small vessel ischemic disease in the brain. Microvesicles (MV) derived from activated cells vary by cell of origin and the stimulus initiating their release. MV released from cells activated by inflammatory and thrombotic factors have the potential to disrupt endothelial cells of the brain microvasculature. Therefore, experiments were designed to identify sex differences in the phenotype of MV released from cultured human brain microvascular endothelial cells (HBMEC) in response to inflammatory and thrombotic stimuli. Methods Cultured HBMEC derived from 20- to 30-year-old male and female donors were treated for 20 h with medium supplemented with tumor necrosis factor alpha (TNFα; 20 ng/ml), thrombin (THR; 2 U/ml), or vehicle (i.e., control). MV were isolated from the conditioned media by high-speed centrifugation and quantified by digital flow cytometry by labeling with fluorophore-conjugated primary antibodies against PECAM-1, integrin αvβ3, ICAM-1, E-selectin, or MCAM. In addition, temporal uptake of labeled MV into control HBMEC was examined by confocal microscopy. Results Under control conditions, male HBMEC released fewer MV expressing each antigen, except for PECAM-1, than female cells ( P < 0.05). Neither TNFα nor THR reduced cell viability. However, TNFα induced apoptosis in female and male cells, whereas THR increased apoptosis marginally only in male cells. TNFα increased expression of all antigens tested on MV in male cells, but only increased expression of integrin αvβ3, ICAM-1, and E-selectin on MV from female cells. THR increased expression of PECAM-1, ICAM-1, and MCAM-1 on MV from male but not female cells. MV were internalized and localized to lysosomes within 90 min after their application to HBMEC. Conclusions There are sex differences in expression of cell adhesion molecules on MV released from HBMEC under control conditions and upon activation by TNFα or THR. MV taken up by unstimulated HBMEC may impact the integrity of the brain microvasculature and account, in part, for sex differences in vascular pathologies in the brain.

Considerations Upon Pterygium Perception in Terms of Symptoms Induced by Inflammatory Response, According to Sex and Age

“…There are numerous studies which emphasize the difference of inflammatory response in males and females for various diseases and conditions [22][23][24], which would explain these results. In fact, it was demonstrated that females tend to have a higher number of leukocytes (more macrophages and more T and B lymphocytes) compared to males, more than double, this difference being explained by various values of tissue chemokines; also, resident leukocytes have a different phenotype in males and females [25].…”

Vesicular Transport of Encapsulated microRNA between Glial and Neuronal Cells

“…EXs and EMVs: (i) are released under normal physiological conditions, but are also discharged from parent cells upon cellular activation, hypoxia and/or hyperoxia, senescence, apoptosis and disease via a paracrine- and endocrine-type type action to their target cells; (ii) represent one of the major biological mechanisms for genetic exchange, immune signaling and the spread of inflammation and disease between cells of the host; (iii) EX and EMV trafficking in the mammalian CNS is a particularly robust, active and dynamic process (Valadi et al, 2007 [ 26 ]; Hunter et al, 2008 [ 36 ]; Deng et al, 2018 [ 34 ]; Stahl et al, 2019 [ 9 ]; Arbo et al, 2020 [ 6 ]; Hou et al, 2020 [ 37 ]). The human brain and retina, CNS, cerebrospinal fluid (CSF), neurovasculature, as well as the systemic circulation, are particularly rich sources of EXs, MPs and EMVs, suggesting that they are significant components of a highly active multi-component extracellular signaling and communication system (Trotta et al, 2018 [ 38 ]; Seyedrazizadeh et al, 2020 [ 39 ]; Song et al, 2020 [ 40 ]).…”

“…The discovery of the important role of miRNAs in the regulation of the transcriptome of a cell was made about ~15 years ago and was shortly followed by the first reports of altered miRNA abundance, speciation and complexity in the limbic system of Alzheimer’s disease (AD) brains (Lukiw 2007 [ 60 ]) This included a significant up-regulation of what are now known as “ pro-inflammatory miRNAs ”, including miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155 and others in the parenchyma of the temporal lobe neocortex (Lukiw 2007 [ 60 ]; Valadi et al, 2007 [ 26 ]; Hunter et al, 2008 [ 36 ]; Sethi and Lukiw 2009 [ 22 ]; Zhao et al, 2015 [ 61 ]; Hammond 2016 [ 28 ]). The first reports of EXs and EMVs being loaded with ribonucleic acid cargoes, such as specific miRNAs and mRNAs, first came about ~12 years ago: (i) from the microarray analysis of mast cells involved in innate and adaptive immunity, autoimmunity, and inflammation (Valadi et al, 2007 [ 26 ]); (ii) from studies of specific miRNAs, mRNAs and angiogenic proteins in glioblastoma and neuroblastoma tumor cells that have released EXs and EMVs (Skog et al, 2008 [ 25 ]; Columbo et al, 2014 [ 13 ]; Briand et al, 2020 [ 8 ]).…”

“…The first reports of EXs and EMVs being loaded with ribonucleic acid cargoes, such as specific miRNAs and mRNAs, first came about ~12 years ago: (i) from the microarray analysis of mast cells involved in innate and adaptive immunity, autoimmunity, and inflammation (Valadi et al, 2007 [ 26 ]); (ii) from studies of specific miRNAs, mRNAs and angiogenic proteins in glioblastoma and neuroblastoma tumor cells that have released EXs and EMVs (Skog et al, 2008 [ 25 ]; Columbo et al, 2014 [ 13 ]; Briand et al, 2020 [ 8 ]). These extruded vesicles were subsequently observed to be taken up by normal host brain microvascular endothelial cells, thereby inducing carcinogenic type phenotypic change in these brain cells, stimulating tumor invasion, proliferation and cancer spread mediated by endothelial cells of the neurovasculature (Hunter et al, 2008 [ 36 ]; Skog et al, 2008 [ 25 ]). Very recently, evidence has been provided showing that EXs and EMVs derived from miRNA-containing natural killer (NK) cells contribute to immunological surveillance and their provision of specific miRNAs may be the first-line of defense in the regulation of tumor cell growth, cytotoxicity and metastasis diffusion in the CNS (Briand et al, 2020 [ 8 ]; Federici et al, 2020 [ 62 ]).…”

Comprehensive phenotyping of endothelial cells using flow cytometry 2: Human

“…Inducible markers used to identify endothelial dysfunction or injury are similar between the human and murine endothelium, with few exceptions. CD62E (E‐selectin) and CD106 (V‐CAM) are human‐specific markers of endothelial damage not used in murine panels [5,18,20,28‐39]. A full list of inducible markers for human endothelial cells can be found in Table 3.…”