Early Passage Endothelial Cells Research Articles

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.

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.

Impact of Senescent Endothelial Cells on Vascular Smooth Muscle Cells

Cellular senescence was at one time thought to be benign, but it is becoming evident that senescent cells are pro-inflammatory and may be associated with other deleterious consequences in aging organisms that accumulate senescent cells over time. In particular, senescent endothelial cells are known to secrete markedly more pro-inflammatory factors than non-senescent cells, and more disturbingly, may also incite pro-inflammatory secretion in the healthy non-senescent cells along with other negative functional changes. To investigate this issue, we co-cultured human microvascular smooth muscle cells (VSMC) with senescent (SEN) or early-passage (EP) human endothelial cells (EC), separated by a permeable membrane to allow both VSMC and EC to communicate with secreted factors. We found that the coculture of VSMC with EC markedly and synergistically elevated secreted pro-inflammatory factors, IL-6, IL-8, and MCP-1. While both SEN and EP cells promoted the cytokine and chemokine release by comparable levels, the amount released in SEN EC – VSMC coculture was 1.5- to 2- fold greater than in EP EC – VSMC co-culture. In addition, the beta 1 subunit of soluble guanylate cyclase, an NO receptor that promotes vasodilation, was decreased in the VSMC from SEN EC – VSMC coculture, but not in EP EC – VSMC coculture. These findings suggest the potential role of senescent EC in aggravating hypertension and persistent inflammatory disorder that are common in older individuals.

High levels of oncomiR‐21 contribute to the senescence‐induced growth arrest in normal human cells and its knock‐down increases the replicative lifespan

Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21CIP1 and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21CIP1, and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.

Clonal senescence alters endothelial ICAM-1 function

Little is known how age alters the dynamics and function of cell adhesion molecules, especially under inflammatory and stressful conditions. One membrane constituent, intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily that regulates key outside → in and inside → out signals associated with cell-to-cell interactions. If conditions such as age and inflammation change usual ICAM-1 action then important downstream effects ultimately perturb endothelial cell function. In this report, ICAM-1 accumulates in late passage endothelial cells when compared to early passage endothelial cells, yet ICAM-1 protein expression is attenuated when senescent cells are challenged by TNF-α (10 ng/ml). Importantly, age alters ICAM-1 dynamic properties from directed to random receptor motion within the membrane. Single particle tracking reveals that the average ICAM-1 mobility is 44% less in late than early passage cells after its motion is stimulated by the Protein Kinase C (PKC) activator, phorbol myristate acetate (PMA). The mechanism for altered ICAM-1 mobility partly can be explained by a reduced rate of alpha-actinin linking with ICAM-1 in late passage Human Pulmonary Artery Endothelial Cells (HPAECs). Furthermore, tyrosine phosphorylation of alpha-actinin, a requirment for ICAM-1 clustering, is markedly reduced in senescent cells. These findings support a hypothesis that senescence results in changes of ICAM-1 activation and clustering, thus resulting in an age-dependent transmembrane signaling disorder. Therefore, further understanding of age-dependent disturbances of ICAM-1 regulation during inflammation can provide important clues as to appropriate targets for therapeutic interventions and prevention of vascular disorders in elderly at the level of the endothelial surface membrane.

Differential responsiveness of early and late passage (SENESCENT) endothelial cells to flow

Hemodynamic forces such as shear stress are implicated in the development of atherosclerosis and neointimal hyperplasia. To determine whether in vitro studies of flow using early passage endothelial cells (EC) are relevant to elderly patients, the effects of flow on early passage and late passage (senescent) EC were compared. Early (p3–7) and late (p28–32) passage bovine aortic EC were exposed to arterial levels of flow (11 dyn/cm 2) using an orbital shaker; control cells were exposed to static conditions (0 dyn/cm 2). Cell proliferation was counted with a Coulter counter. PCNA was evaluated by immunohistochemistry. Phosphorylated and total Akt were assessed with Western blotting. Conditioned media from early and late passage EC under static and flow conditions (16 h) were evaluated for their chemoattractant activities by assessing smooth muscle cell (SMC) migration (4 h, Boyden chamber). Early passage cells demonstrated increased proliferation with flow compared to static conditions (36% increase, day 5, P = 0.03); late passage cells had no statistically significant increase ( P = 0.42). PCNA staining confirmed lack of proliferation in late passage cells under static or flow conditions. Western blotting demonstrated increased Akt phosphorylation with flow in early, but not late, passage EC ( n = 6, P = 0.01). Senescent cells induced 26% less SMC migration than early passage cells ( n = 3, P = 0.03). Late passage EC do not proliferate in response to turbulent flow as younger EC do. Late passage EC also do not phosphorylate Akt, and attract fewer SMC, than early passage EC, in response to flow. These results suggest that hemodynamic forces may have different effects on senescent compared to younger EC and implies that treatment of vascular disease in elderly patients may require different therapeutic strategies than those based on strategies designed for younger patients.

Pro-angiogenic Activities of CYR61 (CCN1) Mediated through Integrins αvβ3 and α6β1 in Human Umbilical Vein Endothelial Cells

CYR61 (CCN1) is an extracellular matrix-associated protein of the CCN family, which also includes CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). Purified CYR61 induces neovascularization in corneal implants, and Cyr61-null mice suffer embryonic death due to vascular defects, thus establishing that CYR61 is an important regulator of angiogenesis. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. In culture, CYR61 functions through integrin-mediated pathways to promote cell adhesion, migration, and proliferation. Here we show that CYR61 can also promote cell survival and tubule formation in human umbilical vein endothelial cells. Furthermore, we have dissected the integrin receptor requirements of CYR61 with respect to its pro-angiogenic activities. Thus, CYR61-induced cell adhesion and tubule formation occur through interaction with integrin alpha(6)beta(1) in early passage endothelial cells in which integrins have not been activated. By contrast, in endothelial cells in which integrins are activated by phorbol ester or vascular endothelial growth factor, CYR61-promoted cell adhesion, migration, survival, growth factor-induced mitogenesis, and endothelial tubule formation are all mediated through integrin alpha(v)beta(3). These findings indicate that CYR61 is an activation-dependent ligand of integrin alpha(v)beta(3) and an activation-independent ligand of integrin alpha(6)beta(1) and that these integrins differentially mediate the pro-angiogenic activities of CYR61. These findings help to define the mechanisms by which CYR61 acts as an angiogenic regulator, provide a molecular interpretation for the loss of vascular integrity and increased apoptosis of vascular cells in Cyr61-null mice, and underscore the importance of CYR61 in the development and homeostasis of the vascular system.

Altered Expression of Cyclic Nucleotide Phosphodiesterase Isozymes during Culture of Aortic Endothelial Cells

Primary cultures of bovine aortic endothelial cells (BAEC) express cyclic nucleotide phosphodiesterase (CN PDE) isozymes of the PDE2, PDE4 and PDE5 gene families. We report here that the isozyme profiles of CN PDE and the amounts of each vary with the passage number of BAEC cultures. Characterization by anion-exchange chromatography and pharmacological criteria were used to study CN PDE in early (4–6), intermediate (6–10), and late (>17) passages of purified BAEC. PDE2 and a minor fraction of PDE5 accounted for cyclic GMP hydrolysis in early passages, but both isozymes were lost with cell passage. Cyclic AMP was hydrolyzed by both PDE2 and PDE4 isozymes in early passage endothelial cells, but PDE4 was increased dramatically in higher passage cells. Also appearing in the higher passage cells were prominent PDE1 and minor PDE3 activities. The ratios of cytosolic to particulate activities were similar at all passages. BAEC PDE isoforms in intact cells assessed by [ 3H]-adenine prelabeling showed that atriopeptin II decreased isoproterenol-induced cyclic AMP accumulation in early but not later passage cells, consistent with the loss of PDE2 expression. Enhancement of isoproterenol-induced cyclic AMP accumulation by rolipram, a PDE4 inhibitor, was also greatly diminished during culture passages. Changes in CN PDE isoform expression and consequent cyclic AMP turnover validate the importance of considering cell passage number when cultures of BAEC are used to study the regulation of endothelial cell cyclic nucleotide metabolism and processes mediated by cyclic nucleotides in this model system.

Inhibition of Proteoglycan Synthesis in Human Endothelial Cells after Infection with Herpes Simplex Virus Type 1 In Vitro

The effects of herpes simplex virus type 1 (HSV-1) infection on proteoglycan synthesis by human endothelial cells were studied as a model of endothelial cell injury. Confluent cultures of early passage endothelial cells from human umbilical vein were infected with HSV-1 at multiplicities of infection from 0.001 to 1.0. HSV-1 infection produced a dose-dependent inhibition of total proteoglycan synthesis of up to 85%. Although there was a 2- to 3-fold increase in the quantity of virus necessary to cause 50% inhibition of heparan sulfate compared to chondroitin/dermatan sulfate proteoglycan, the inhibition was relatively parallel, even up to high virus doses. There was no inhibition of an undersulfated heparan sulfate proteoglycan that contained glycosaminoglycan chains shorter than the predominant species. The results indicate that HSV-1 infection of human endothelial cells produces complex effects on host-cell metabolism. The viral-induced changes in proteoglycan metabolism may influence cell-matrix interactions and lead to altered vessel wall function.

Differences in secretion of prostacyclin by venous and arterial endothelial cells grown in vitro in a static versus a mechanically active environment

The objective of this study was to compare the effect of cyclic tension on prostacyclin secretion by venous and arterial endothelial cells. Early passage endothelial cells from bovine aortas and venae cavae were subjected to cyclic deformation (up to 17% elongation and 60 cycles/min). On posttreatment days 3 and 5 a radioimmunoassay was used to assess supernatant fluids from the endothelial cells for prostacyclin activity. The results indicate that in vitro (1) under static or control conditions, venous endothelial cells secrete significantly more prostacyclin (an increase of 1.5- and 4.8-fold on days 3 and 5, respectively) than do arterial endothelial cells isolated from the same animal and (2) prostacyclin secretion by mechanically deformed venous and arterial endothelial cells was significantly less than static control cultures on days 3 and 5. However, prostaglandin I2 secretion remained at higher levels in cyclically deformed venous endothelial cells than in cyclically deformed arterial endothelial cells. These data suggest that endothelial cells from the vena cava have a greater capacity for prostacyclin secretion than have their aortic counterparts. If these observations are maintained in vivo, greater prostacyclin secretion by venous endothelial cells could represent a homeostatic mechanism aimed at reducing thrombus formation in low-velocity areas of the vasculature.