Early Endothelial Cells Research Articles

Endothelial cell transitions in zebrafish vascular development.

In recent decades, developmental biologists have come to view vascular development as a series of progressive transitions. Mesoderm differentiates into endothelial cells; arteries, veins and lymphatic endothelial cells are specified from early endothelial cells; and vascular networks diversify and invade developing tissues and organs. Our understanding of this elaborate developmental process has benefitted from detailed studies using the zebrafish as a model system. Here, we review a number of key developmental transitions that occur in zebrafish during the formation of the blood and lymphatic vessel networks.

Transcriptome analysis of cardiac endothelial cells after myocardial infarction reveals temporal changes and long-term deficits.

Endothelial cells (ECs) have essential roles in cardiac tissue repair after myocardial infarction (MI). To establish stage-specific and long-term effects of the ischemic injury on cardiac ECs, we analyzed their transcriptome at landmark time points after MI in mice. We found that early EC response at Day 2 post-MI centered on metabolic changes, acquisition of proinflammatory phenotypes, initiation of the S phase of cell cycle, and activation of stress-response pathways, followed by progression to mitosis (M/G2 phase) and acquisition of proangiogenic and mesenchymal properties during scar formation at Day 7. In contrast, genes involved in vascular physiology and maintenance of vascular tone were suppressed. Importantly, ECs did not return to pre-injury phenotypes after repair has been completed but maintained inflammatory, fibrotic and thrombotic characteristics and lost circadian rhythmicity. We discovered that the highest induced transcript is the mammalian-specific Sh2d5 gene that promoted migration and invasion of ECs through Rac1 GTPase. Our results revealed a synchronized, temporal activation of disease phenotypes, metabolic pathways, and proliferation in quiescent ECs after MI, indicating that precisely-timed interventions are necessary to optimize cardiac tissue repair and improve outcomes. Furthermore, long-term effects of acute ischemic injury on ECs may contribute to vascular dysfunction and development of heart failure.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

Meis1 establishes the pre-hemogenic endothelial state prior to Runx1 expression

Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. Characterization of early hemogenic endothelial (HE) cells is required to understand what drives hemogenic specification and to accurately define cells capable of undergoing EHT. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we define the early subpopulation of pre-HE cells based on both surface markers and transcriptomes. We identify the transcription factor Meis1 as an essential regulator of hemogenic cell specification in the embryo prior to Runx1 expression. Meis1 is expressed at the earliest stages of EHT and distinguishes pre-HE cells primed towards the hemogenic trajectory from the arterial endothelial cells that continue towards a vascular fate. Endothelial-specific deletion of Meis1 impairs the formation of functional Runx1-expressing HE which significantly impedes the emergence of pre-HSPC via EHT. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

ETV2 Upregulation Marks the Specification of Early Cardiomyocytes and Endothelial Cells During Co-differentiation.

The ability to differentiate human-induced pluripotent stem cells (hiPSCs) efficiently into defined cardiac lineages, such as cardiomyocytes and cardiac endothelial cells, is crucial to study human heart development and model cardiovascular diseases in vitro. The mechanisms underlying the specification of these cell types during human development are not well understood which limits fine-tuning and broader application of cardiac model systems. Here, we used the expression of ETV2, a master regulator of hematoendothelial specification in mice, to identify functionally distinct subpopulations during the co-differentiation of endothelial cells and cardiomyocytes from hiPSCs. Targeted analysis of single-cell RNA-sequencing data revealed differential ETV2 dynamics in the 2 lineages. A newly created fluorescent reporter line allowed us to identify early lineage-predisposed states and show that a transient ETV2-high-state initiates the specification of endothelial cells. We further demonstrated, unexpectedly, that functional cardiomyocytes can originate from progenitors expressing ETV2 at a low level. Our study thus sheds light on the in vitro differentiation dynamics of 2 important cardiac lineages.

Senescent endothelial cells are predisposed to SARS-CoV-2 infection and subsequent endothelial dysfunction

The coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains to spread worldwide. COVID-19 is characterized by the striking high mortality in elderly; however, its mechanistic insights remain unclear. Systemic thrombosis has been highlighted in the pathogenesis of COVID-19, and lung microangiopathy in association with endothelial cells (ECs) injury has been reported by post-mortem analysis of the lungs. Here, we experimentally investigated the SARS-CoV-2 infection in cultured human ECs, and performed a comparative analysis for post-infection molecular events using early passage and replicative senescent ECs. We found that; (1) SARS-CoV-2 infects ECs but does not replicate and disappears in 72 hours without causing severe cell damage, (2) Senescent ECs are highly susceptible to SARS-CoV-2 infection, (3) SARS-CoV-2 infection alters various genes expression, which could cause EC dysfunctions, (4) More genes expression is affected in senescent ECs by SARS-CoV-2 infection than in early passage ECs, which might causes further exacerbated dysfunction in senescent ECs. These data suggest that sustained EC dysfunctions due to SARS-CoV-2 infection may contribute to the microangiopathy in the lungs, leading to deteriorated inflammation and thrombosis in COVID-19. Our data also suggest a possible causative role of EC senescence in the aggravated disease in elder COVID-19 patients.

Endothelial dysfunction aggravates arterial media calcification in warfarin administered rats.

Arterial media calcification is an active cell process. This encompasses osteochondrogenic transdifferentiation of vascular smooth muscle cells followed by the deposition of calcium-phosphate crystals. Increasing evidence suggests a significant role for endothelial cells (ECs) in the development of arterial media calcification. This manuscript explores a role for endothelial dysfunction in the disease progression of arterial media calcification. Male rats were randomly assigned to four different groups. The first group received standard chow. The second group was given L-NAME (≈50mg kg-1 · d-1 ), to induce endothelial dysfunction, in addition to standard chow. The third group and fourth group received a warfarin-supplemented diet to induce mild calcification and the latter group was co-administered L-NAME. Prior to sacrifice, non-invasive measurement of aortic distensibility was performed. Animals were sacrificed after 6weeks. Arterial media calcification was quantified by measuring aortic calcium and visualized on paraffin-embedded slices by the Von Kossa method. Arterial stiffness and aortic reactivity was assessed on isolated carotid segments using specialized organ chamber setups. Warfarin administration induced mineralization. Simultaneous administration of warfarin and L-NAME aggravated the arterial media calcification process. Through organ chamber experiments an increased vessel tonus was found, which could be linked to reduced basal NO availability, in arteries of warfarin-treated animals. Furthermore, increased calcification because of L-NAME administration was related to a further compromised endothelial function (next to deteriorated basal NO release also deteriorated stimulated NO release). Our findings suggest early EC changes to impact the disease progression of arterial media calcification.

Tetrahedral Framework Nucleic Acids Connected with MicroRNA-126 Mimics for Applications in Vascular Inflammation, Remodeling, and Homeostasis.

The repair of damaged endothelium is crucial for vascular homeostasis maintenance, which comprises the recovery of early stage impaired endothelial cells and migration of surrounding unimpaired endothelial cells. MicroRNAs (miRNAs) play an indispensable role in balancing gene expression in organisms. For vascular tissues, miR-126 is one of the most important regulators and might have substantial application potential in maintaining vascular homeostasis. In this study, a type of sticky-end-modified tetrahedral framework nucleic acids (tFNAs-SE) was employed to successfully link the miR-126 5p mimic duplex, which was termed tFNAs-miR-126 5p mimics (tFNAs-MMs). Existing vascular endothelial growth factors (VEGF), tFNAs-MMs can improve cell viability, resist apoptosis, and recover the state and functions of LPS-induced impaired human umbilical vein endothelial cells (HUVECs). The angiogenesis ability of impaired HUVECs was recovered by tFNAs-MMs in vitro and in vivo. The mechanisms underlying these phenomena were demonstrated to be related to the downregulation of caspase3 and negative regulators of VEGF (SPRED1 and PIK3R2). Moreover, tFNAs-MMs promoted the migration and proliferation of HUVECs. Briefly, the strategy of sticky-end-modified tFNAs connecting miRNA mimics is available for miRNA gain of function, while tFNAs-MMs might be a promising agent for repairing early stage vascular damage and maintaining vascular homeostasis.

Pre-configuring chromatin architecture with histone modifications guides hematopoietic stem cell formation in mouse embryos

The gene activity underlying cell differentiation is regulated by a diverse set of transcription factors (TFs), histone modifications, chromatin structures and more. Although definitive hematopoietic stem cells (HSCs) are known to emerge via endothelial-to-hematopoietic transition (EHT), how the multi-layered epigenome is sequentially unfolded in a small portion of endothelial cells (ECs) transitioning into the hematopoietic fate remains elusive. With optimized low-input itChIP-seq and Hi-C assays, we performed multi-omics dissection of the HSC ontogeny trajectory across early arterial ECs (eAECs), hemogenic endothelial cells (HECs), pre-HSCs and long-term HSCs (LT-HSCs) in mouse embryos. Interestingly, HSC regulatory regions are already pre-configurated with active histone modifications as early as eAECs, preceding chromatin looping dynamics within topologically associating domains. Chromatin looping structures between enhancers and promoters only become gradually strengthened over time. Notably, RUNX1, a master TF for hematopoiesis, enriched at half of these loops is observed early from eAECs through pre-HSCs but its enrichment further increases in HSCs. RUNX1 and co-TFs together constitute a central, progressively intensified enhancer-promoter interactions. Thus, our study provides a framework to decipher how temporal epigenomic configurations fulfill cell lineage specification during development.

Potential contribution of early endothelial progenitor cell (eEPC)-to-macrophage switching in the development of pulmonary plexogenic lesion

BackgroundPlexiform lesions, which have a dynamic appearance in structure and cellular composition, are the histological hallmark of severe pulmonary arterial hypertension in humans. The pathogenesis of the lesion development remains largely unknown, although it may be related to local inflammation and dysfunction in early progenitor endothelial cells (eEPCs). We tested the hypothesis that eEPCs contribute to the development of plexiform lesions by differentiating into macrophages in the setting of chronic inflammation.MethodsThe eEPC markers CD133 and VEGFR-2, macrophage lineage marker mannose receptor C-type 1 (MRC1), TNFα and nuclear factor erythroid 2-related factor 2 (Nrf2) in plexiform lesions in a broiler model were determined by immunohistochemistry. eEPCs derived from peripheral blood mononuclear cells were exposed to TNFα, and macrophage differentiation and angiogenic capacity of the cells were evaluated by phagocytotic and Matrigel plug assays, respectively. The role of Nrf2 in eEPC-to-macrophage transition as well as in MRC1 expression was also evaluated. Intratracheal installation of TNFα was conducted to determine the effect of local inflammation on the formation of plexiform lesions.ResultsCells composed of the early lesions have a typical eEPC phenotype whereas those in more mature lesions display molecular and morphological characteristics of macrophages. Increased TNFα production in plexiform lesions was observed with lesion progression. In vitro studies showed that chronic TNFα challenge directed eEPCs to macrophage differentiation accompanied by hyperactivation of Nrf2, a stress-responsive transcription factor. Nrf2 activation (Keap1 knockdown) caused a marked downregulation in CD133 but upregulation in MRC1 mRNA. Dual luciferase reporter assay demonstrated that Nrf2 binds to the promoter of MRC1 to trigger its expression. In good agreement with the in vitro observation, TNFα exposure induced macrophage differentiation of eEPCs in Matrigel plugs, resulting in reduced neovascularization of the plugs. Intratracheal installation of TNFα resulted in a significant increase in plexiform lesion density.ConclusionsThis work provides evidence suggesting that macrophage differentiation of eEPCs resulting from chronic inflammatory stimulation contributes to the development of plexiform lesions. Given the key role of Nrf2 in the phenotypic switching of eEPCs to macrophages, targeting this molecular might be beneficial for intervention of plexiform lesions.

PEA prevented early BBB disruption after cerebral ischaemic/reperfusion (I/R) injury through regulation of ROCK/MLC signaling

Palmitoylethanolamide (PEA) offers a strong protection against BBB disruption and neurological deficits after cerebral ischaemic/reperfusion (I/R) injury. To date, these BBB protective effects of PEA are mainly attributed to PPARα-mediated actions. However, whether PEA protects against BBB disruption through direct regulation of cytoskeletal microfilaments remains unknown. Here, we identified PEA as a Rho-associated protein kinase (ROCK2) inhibitor (IC50 = 38.4 ± 4.8 μM). In vitro data suggested that PEA reduced the activation of ROCK/MLC signaling and stress fiber formation within microvascular endothelial cells (ECs) after oxygen-glucose deprivation (OGD), and consequently attenuated early (0–4 h) EC barrier disruption. These actions of PEA could not be blocked by the PPARα antagonist GW6471. In summary, the present study described a previously unexplored role of PEA as a ROCK2 inhibitor, and propose a PPARα-independent mechanism for pharmacological effects of PEA.

A mouse model for kinesin family member 11 (Kif11)-associated familial exudative vitreoretinopathy.

During mitosis, Kif11, a kinesin motor protein, promotes bipolar spindle formation and chromosome movement, and during interphase, Kif11 mediates diverse trafficking processes in the cytoplasm. In humans, inactivating mutations in KIF11 are associated with (1) retinal hypovascularization with or without microcephaly and (2) multi-organ syndromes characterized by variable combinations of lymphedema, chorioretinal dysplasia, microcephaly and/or mental retardation. To explore the pathogenic basis of KIF11-associated retinal vascular disease, we generated a Kif11 conditional knockout (CKO) mouse and investigated the consequences of early postnatal inactivation of Kif11 in vascular endothelial cells (ECs). The principal finding is that postnatal EC-specific loss of Kif11 leads to severely stunted growth of the retinal vasculature, mildly stunted growth of the cerebellar vasculature and little or no effect on the vasculature elsewhere in the central nervous system (CNS). Thus, in mice, Kif11 function in early postnatal CNS ECs is most significant in the two CNS regions-the retina and cerebellum-that exhibit the most rapid rate of postnatal growth, which may sensitize ECs to impaired mitotic spindle function. Several lines of evidence indicate that these phenotypes are not caused by reduced beta-catenin signaling in ECs, despite the close resemblance of the Kif11 CKO phenotype to that caused by EC-specific reductions in beta-catenin signaling. Based on prior work, defective beta-catenin signaling had been the only known mechanism responsible for monogenic human disorders of retinal hypovascularization. The present study implies that retinal hypovascularization can arise from a second and mechanistically distinct cause.

Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage.

Breakdown of blood-brain barrier, formed mainly by brain microvascular endothelial cells (BMECs), represents the major cause of mortality during early phases of ischemic strokes. Hence, discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine. Although endothelial progenitor cells (EPCs) represent one such agents, their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells (OECs) as a result. Functional analyses of these cells, in the present study, demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel, prominent endothelial cell and angiogenesis markers, respectively. Further analyses by flow cytometry demonstrated that OECs expressed specific markers for stemness (CD34), immaturity (CD133) and endothelial cells (CD31) but not for hematopoietic cells (CD45). Like BMECs, OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes, suggesting their capacity to form tight junctions. Ischemic injury mimicked by concurrent deprivation of oxygen and glucose (4 hours) or deprivation of oxygen and glucose followed by reperfusion (20 hours) affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux, respectively. Wound scratch assays comparing the vasculoreparative capacity of cells revealed that, compared to BMECs, OECs possessed a greater proliferative and directional migratory capacity. In a triple culture model of BBB established with astrocytes, pericytes and BMEC, exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions. Taken together, these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain (neuro)vascular homeostasis during or after a cerebral ischemic injury.

MicroRNA-126 regulates Hypoxia-Inducible Factor-1\u03b1 which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence

Whereas a healthy endothelium maintains physiological vascular functions, endothelial damage contributes to the development of cardiovascular diseases. Endothelial senescence is the main determinant of endothelial dysfunction and thus of age-related cardiovascular disease. The objective of this study is to test the involvement of microRNA-126 and HIF-1α in a model of replicative endothelial senescence and the interrelationship between both molecules in this in vitro model. We demonstrated that senescent endothelial cells experience impaired tube formation and delayed wound healing. Senescent endothelial cells failed to express HIF-1α, and the microvesicles released by these cells failed to carry HIF-1α. Of note, HIF-1α protein levels were restored in HIF-1α stabilizer-treated senescent endothelial cells. Finally, we show that microRNA-126 was downregulated in senescent endothelial cells and microvesicles. With regard to the interplay between microRNA-126 and HIF-1α, transfection with a microRNA-126 inhibitor downregulated HIF-1α expression in early passage endothelial cells. Moreover, while HIF-1α inhibition reduced tube formation and wound healing closure, microRNA-126 levels remained unchanged. These data indicate that HIF-1α is a target of miRNA-126 in protective and reparative functions, and suggest that their therapeutic modulation could benefit age-related vascular disease.

Aged endothelial cells exhibit a metabolic shift from anaerobic glycolysis to oxidative phosphorylation

Endothelial dysfunction is a hallmark of vascular aging and is associated with increased arterial oxidative stress. Mitochondria, via the electron transport chain, are thought to be an important source of cellular superoxide that suppresses endothelial function in older humans but surprisingly little is understood about the effect of aging on endothelial cell metabolic activity. Young, normally functioning endothelial cells typically utilize anaerobic glycolytic metabolism but with aging a shift toward a greater reliance on oxidative metabolism could explain enhanced superoxide production via the electron transport chain. Here we aimed to determine the impact of aging on endothelial cell metabolism. To do so, we utilized SeahorseTM technology to assess mitochondrial oxygen consumption and extracellular acidification rate as measures of oxidative and anaerobic glycolytic metabolism in primary lung endothelial cells (ECs) isolated from young (4–6M) and old (16–18M) C57BL/6 mice, as well as in early (P3) and late (P16) passage human umbilical vein endothelial cells (HUVECs), a cell culture model of aging. We found that old primary ECs demonstrate a 37% higher rate of oxygen consumption (p<0.01), 74% higher spare respiration (p<0.01) and 92% higher proton leak (p<0.01) compared with young primary ECs. In contrast, ATP production was 27% lower in old compared with young primary ECs (p<0.01). Total extracellular acidification rate (glycolytic shift) was 47% (p<0.01) lower and maximal glycolytic rate was 22% lower (p<0.01) in old compared with young primary ECs, indicating reduced glycolysis and increased oxidative phosphorylation in old ECs. Next, we assessed mitochondrial membrane potential, pyruvate kinase (PK) activity, lactate dehydrogenase (LDH) activity, as well as the expression of mitochondrial membrane complexes by western blot. Interestingly, we observed 62% lower mitochondrial membrane potential (p<0.05), and reduced expression of mitochondrial membrane complex II (34%, p<0.05), III (65%, p<0.01), IV (87%, p<0.01) and V (56%, p<0.01) in old compared with young ECs. PK activity was increased by 62% (p<0.01) and LDH activity was decreased by 72% (p<0.01) in old compared with young ECs. Similar differences were observed between early and late passage HUVECs. Taken together, these findings suggest that aging results in a shift away from anaerobic glycolysis and towards a greater reliance on oxidative phosphorylation in old ECs. This is despite less efficient mitochondrial coupling and lower protein expression of mitochondrial electron transport chain proteins. Overall, these results suggest that this age related metabolic shift in ECs may contribute to increased mitochondrial superoxide production and subsequent endothelial dysfunction.Support or Funding InformationSupported by awards from the National Institute of Health (R01 AG040297, R01 AG048366, K02 AG045339) and US Department of Veterans Affairs (1I01BX002151).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

SummaryExogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

The relationship between number and function of EPCs and concentration of VEGF165 and SDF-1 in coronary artery spasm.

We aimed at investigating the functions of endothelial progenitor cells (EPCs) in the pathophysiology of coronary artery spasm (CAS) and coronary artery disease (CAD), as well as to evaluate the correlation of these diseases with the number and function of EPCs, the plasma concentration of vascular endothelial growth factor 165 (VEGF165) and stromal cell-derived factor 1 (SDF-1). Participants were recruited into three groups, CAS, CAD and the control. The number and functions of early EPCs and outgrowth endothelial cells (OECs) were determined in peripheral blood samples, and the endothelial function was evaluated by measuring endothelium-dependent flow-mediated vasodilation (FMD). No differences of baseline characteristics were found among CAS, CAD, and the control groups. The OECs isolated from CAS and CAD exhibited significant decrease in the percentage of CD34+/CD45- population, OEC colony formation, OEC proliferation and OEC tubulogenesis, nitric oxide (NO) production, endothelial nitric oxide synthase (eNOS) activity, and the phosphorylation level at Ser1177 of eNOS, compared with OECs isolated from control participants. Meanwhile, FMD was significantly reduced in CAS and CAD (CAS, 4.1% ± 1.9%; CAD, 4.3% ± 1.8%; control, 11.2% ± 3.5%). FMD was positively correlated to OEC functions including NO production, eNOS phosphorylation, colony formation, and proliferation. No differences of plasma VEGF165 and SDF-1 concentrations were recorded among these three groups. Similarly, there was no correlation between plasma VEGF165 (and SDF-1) concentration and EPC number and function. EPCs show the potential of repairing damaged endothelium in CAS and CAD.

Sulfur mustard-induced epigenetic modifications over time − a pilot study

The chemical warfare agent sulfur mustard (SM) can cause long-term health effects that may occur even years after a single exposure. The underlying pathophysiology is unknown, but epigenetic mechanisms are discussed as feasible explanation. “Epigenetics” depicts regulation of gene function without affecting the DNA sequence itself. DNA-methylation and covalent histone modifications (methylation or acetylation) are regarded as important processes. In the present in vitro study using early endothelial cells (EEC), we analyzed SM-induced DNA methylation over time and compared results to an in vivo skin sample that was obtained approx. one year after an accidental SM exposure. EEC were exposed to low SM concentrations (0.5 and 1.0μM). DNA methylation and histone acetylation (H3-K9, H3-K27, H4-K8) or histone di-methylation (H3-K9, H3-K27, H3-K36) were investigated 24h after exposure, and after 2 or 4 additional cell passages. The human skin sample was assessed in parallel. SM had only some minor effects on histone modifications. However, a significant and pronounced increase of DNA methylation was detected in the late cell passages as well as in the skin sample. Our results indicate that SM does indeed cause epigenetic modifications that appear to persist over time.

STEM/PROGENITOR CELLS ADHESION STRENGTHENING TO SYNTHETIC MATERIAL USING EXTRACELLULAR MATRIX

Background: Tissue-engineered vascular grafts of small diameter have being widely used in coronary artery bypass grafting. However, the rate of settling of these grafts with endothelial cells is insufficient. Aims:The aim of the study was to selucidate the adhesion of endothelial progenitor cells and mesenchymal stem cells to synthetic materials (polystyrol, polytetrafluoroethylene) preprocessed with extracellular matrix (gelatin, fibronectin, collagen) in vitro.Materials and methods: For the study,the endothelial progenitor cells (EPC) were isolated from the peripheral blood of patients with ischemic heart disease, and mesenchymal stem cells (MSC) — from bone marrow of Wistar rats. Commercial monoclonal antibodies for flow- cytometry were used to determine the phenotype of EPC. To isolate the early and late EPC from mononuclear cells of peripheral blood, cells were raised for 8 and 16 days on a gelatin or fibronectin based substrate. The commercial kits for enzyme linked immunoassay were applied to assess levels of cytokine production and nitric oxide by early and late EPC conditioned by gelatin or fibronectin on the 8th and 16th days of growth. To conduct the study the MSC were isolated from bone marrow of rats. To determine the attachment of adherent fraction of nucleated bone marrow cells, cell morphology, the adipogenic and osteogenic differentiation were evaluated. To assess migration of MSC in real time within 24 hours on the device Cell-IQ, we used «closure/wound healing» test. The commercial kits for enzyme linked immunoassay were applied to assess levels of cytokine production and nitric oxide by MSC; and components of the extracellular matrix (fibronectin, collagen I and type IV) ― to assess the increased adhesion of MSC to the polytetrafluoroethylene. Results: The results demonstrated that endothelial progenitor cells adhere to both gelatin and fibronectin and confirmed the influence of these extracellular matrix components on the cytokine levels produced by early and late endothelial cells. The combination of fibronectin with type I or IV collagen or the combination of thereof promotes the adhesion to polytetrafluoroethylene and colonization of the graft.Conclusions: Preprocessing of synthetic material (polystyrene, polytetrafluoroethylene) enhances adhesion and growth of EPC and MSC which can be implemented when creating tissue-engineered vascular grafts for small diameter coronary artery bypass grafting with specified conditions of settlement by the cells involved in neointima formation.