Cardiovascular disease (CVD) is the leading cause of death worldwide, and extensive research has been performed to understand this disease better, using various experimental models. The endothelium ...plays a crucial role in the development of CVD, since it is an interface between bloodstream components, such as monocytes and platelets, and other arterial wall components. Human umbilical vein endothelial cell (HUVEC) isolation from umbilical cord was first described in 1973. To date, this model is still widely used because of the high HUVEC isolation success rate, and because HUVEC are an excellent model to study a broad array of diseases, including cardiovascular and metabolic diseases. We here review the history of HUVEC isolation, the HUVEC model over time, HUVEC culture characteristics and conditions, advantages and disadvantages of this model and finally, its applications in the area of cardiovascular diseases.
Extracellular vesicles (EVs) such as exosomes are nano-sized vesicles that carry proteins and miRNAs and can transmit signals between cells. We hypothesized that exosomes from endothelial cells can ...transmit protective signals to cardiomyocytes. Co-culture of primary adult rat cardiomyocytes with normoxic HUVEC cells separated by a cell-impermeable membrane reduced the percentage of cardiomyocyte death following simulated ischaemia and reperfusion (sIR) from 80 ± 11% to 51 ± 4% (P < 0.05; N = 5). When EVs were removed from the HUVEC-conditioned medium it was no longer protective. Exosomes were purified from HUVEC-conditioned medium using differential centrifugation and characterized by nanoparticle tracking analysis, electron microscopy, and flow cytometry. Pre-incubation of cardiomyocytes with HUVEC exosomes reduced the percentage of cell death after sIR from 88 ± 4% to 55 ± 3% (P < 0.05; N = 3). This protection required ERK1/2 activity as it was prevented by inhibitors PD98059 and U0126. Ischaemic preconditioning caused about ~3-fold higher rate of exosome production from HUVEC and from isolated, perfused rat hearts. This increase resulted in significantly greater protection against sIR in cardiomyocytes. In conclusion, exosomes released from endothelial cells can confer resistance to sIR injury in cardiomyocytes via the activation of the ERK1/2 MAPK signalling pathway, and may contribute to IPC.
TGF‐β induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF‐β1 is enhanced ...by hypoxia and by overexpression of Smad3/4 and hypoxia‐inducible factor‐1α/β (HIF‐1α/β). To examine the transcriptional regulation of VEGF by TGF‐β1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF‐1α/β or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF‐β1, whereas cotransfection of HIF‐1α/β and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF‐1 and two Smad‐binding elements were critical for TGF‐β1‐induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF‐1α/β and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF‐β1 also increased the expression of fetal liver kinase‐1 (Flk‐1), a major VEGF receptor, and TGF‐β1 and VEGF stimulated pro‐matrix metalloproteinase 9 (MMP‐9) and active‐MMP‐9 expression, respectively. The results from the present study indicate that TGF‐β1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP‐9, and Flk‐1.
Endothelial cell death is linked to vascular diseases such as atherosclerosis and tissue ischemia. miRNA-17-92 (miR-17–92) is a multiple functional oncogenic miRNA cluster which plays vital roles in ...tumor angiogenesis and tissue development. However, its role in regulation of endothelial cell ferroptosis remains unclear. In this study, we revealed that miR-17–92 protects endothelial HUVEC cells from erastin-induced ferroptosis. miR-17-92 overexpression significantly reduced erastin-induced growth inhibition and ROS generation of HUVEC cells. Furthermore, Zinc lipoprotein A20, a validated target of miR-17-92, was identified as a novel regulator of endothelial cell ferroptosis. Lentivirus mediated A20 overexpression increased ROS generation and enhanced erastin-induced ferroptosis, whereas A20 knockdown inhibited erastin-induced ferroptosis. Mechanistic studies revealed that erastin-induced ferroptosis is associated with GPX4 downregulation and ACSL4 upregulation. miR-17-92 overexpression or A20 inhibition increased the ACSL4 expression in HUVEC cells. A20 was identified to directly with and regulate ACSL4 expression by immunoprecipitation. It suggests that the A20-ACSL4 axis plays important roles in erastin-induced endothelial ferroptosis. In conclusion, this study revealed a novel mechanism through which miR-17-92 protects endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis.
•miR-17-92 protects endothelial cells from erastin-induced ferroptosis.•Zinc lipoprotein A20 is identified as a novel regulator of ferroptosis.•A20 regulates ACSL4 by their directly interaction in endothelial cells.•miR-17-92 targets the A20-ACSL4 axis in endothelial cells.
Angiogenesis is a hallmark for cancer development because it is essential for cancer growth and provides the route for cancer cell migration (metastasis). Understanding the mechanism of angiogenesis ...and developing drugs that target the process has therefore been a major focus for research on cancer therapy. In this study, we screened 114 FDA-approved anti-cancer drugs for their effects on angiogenesis in the zebrafish. Among those with positive effects, we chose to focus on Ponatinib (AP24534; Iclusig
) for further investigation. Ponatinib is an inhibitor of the tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), and its clinical trial has been approved by FDA for the treatment of the disease. In recent clinical trials, however, some side effects have been reported for Ponatinib, mostly on blood vessel disorders, raising the possibility that this drug may influence angiogenesis. In this study, we demonstrated that Ponatinib was able to suppress the formation of intersegmental vessels (ISV) and subintestinal vessels (SIV) in the zebrafish larvae. The anti-angiogenic effect of Ponatinib was further validated by other bioassays in human umbilical vein endothelial cells (HUVECs), including cell proliferation and migration, tube formation, and wound healing. Further experiments showed that Ponatinib inhibited VEGF-induced VEGFR2 phosphorylation and its downstream signaling pathways including Akt/eNOS/NO pathway and MAPK pathways (ERK and p38MAPK). Taken together, these results suggest that inhibition of VEGF signaling at its receptor level and downstream pathways may likely be responsible for the antiangiogenic activity of Ponatinib.
The development of efficient contrast agents for cell labelling coupled with powerful medical imaging techniques is of great interest for monitoring cell trafficking with potential clinical ...applications such as organ repair and regenerative medicine. In this paper, functionalised multi-walled carbon nanotubes (MWNTs) were engineered for cell labelling in T1-weighted MRI applications. These sophisticated constructs were covalently functionalised with the gadolinium (Gd) chelating agent, diethylene triamine pentaacetic acid (DTPA), enabling tight attachment of Gd atoms onto the nanotube surface. The resulting Gd-labelled MWNTs were found to be stable over 2weeks in water and mouse serum and high payload of Gd atoms could be loaded onto the nanotubes. The r1 relaxivity of the Gd-MWNTs was a 3-fold higher than of the clinically approved T1 contrast agent Magnevist at a magnetic field strength of 7T. The contrast efficiency, expressed as the r1 relaxivity, of the Gd-MWNTs in Human Umbilical Vein Endothelial cells (HUVEC) was investigated at 7T and was found to be around 6.6mM−1s−1. There was no reduction of the contrast efficiency after internalisation in HUVECs, which was imparted to the ability of carbon nanotubes to translocate the cell membrane.
The leading cause of cancer-related death is lung cancer, with metastasis being the most common cause of death. To elucidate the role of macrophages in lung cancer and angiogenesis processes, we ...established an in vitro co-culture model of A549 or HUVEC with THP-1 cells that polarized to M2c macrophages with hydrocortisone. The proteasome inhibitors bortezomib and ixazomib were investigated for their effects on proliferation, invasion, migration, metastasis, and angiogenesis pathways. The effects of bortezomib and ixazomib on gene expression in gene panels, including crucial genes related to angiogenesis and proteasomes, were investigated after the co-culture model to determine these effects at the molecular level. In conclusion, bortezomib and ixazomib showed antiproliferative effects in both cells, as well as in M2c macrophage co-culture. M2c macrophages also increased invasion in A549 cells and both invasion and migration in HUVEC. mRNA expression upregulation, specifically in the NFKB and VEGF genes, supported the metastatic and angiogenic effects found in A549 and HUVEC with M2c macrophage co-culture. Additionally, bortezomib inhibited the VEGFB pathway in HUVEC and NFKB1 in A549 cells. The significant findings obtained as a result of this study will provide information regarding angiogenesis induced by M2 macrophages.
A purified spike (S) glycoprotein of severe acute respiratory syndrome-related coronavirus 2 (SARS
-
CoV
-
2) coronavirus was used to study its effects on THP-1 macrophages, peripheral blood ...mononuclear cells (PBMCs), and HUVEC cells. The S protein mediates the entry of SARS-CoV-2 into cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptors. We measured the viability, intracellular cytokine release, oxidative stress, proinflammatory markers, and THP-1-like macrophage polarization. We observed an increase in apoptosis, ROS generation, MCP-1, and intracellular calcium expression in the THP-1 macrophages. Stimulation with the S protein polarizes the THP-1 macrophages towards proinflammatory futures with an increase in the TNFα and MHC-II M1-like phenotype markers. Treating the cells with an ACE inhibitor, perindopril, at 100 µM reduced apoptosis, ROS, and MHC-II expression induced by S protein. We analyzed the sensitivity of the HUVEC cells after the exposure to a conditioned media (CM) of THP-1 macrophages stimulated with the S protein. The CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, the direct stimulation of the HUVEC cells with the S protein, slightly increased HIF1α and MCP-1 expression, which was significantly increased by the ACE inhibitor treatment. The S protein stimulation induced ROS generation and changed the mitogenic responses of the PBMCs through the upregulation of TNFα and interleukin (IL)-17 cytokine expression. These effects were reduced by the perindopril (100 µM) treatment. Proteomic analysis of the S protein stimulated THP-1 macrophages with or without perindopril (100 µM) exposed more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that the blood and vascular components could be activated directly through S protein systemically present in the circulation and that the activation of the local renin angiotensin system may be partially involved in this process.
Graphical
Suggested pathways that might be involved at least in part in S protein inducing activation of inflammatory markers (red narrow) and angiotensin-converting enzyme inhibitor (ACEi) modulation of this process (green narrow).
Oxidative stress causes endothelial dysfunction, which is associated with vascular cellular aging and is causally related to cardiovascular disease pathogenesis. Preclinical studies indicate that a ...nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide mononucleotide (NMN), alleviates oxidative stress in aged vessels, granting vasoprotective effects. However, the associated cellular mechanism remains largely unclear. In this study, we used human umbilical vein endothelial cells (HUVECs) to demonstrate that NMN inhibits oxidative stress-induced damage by activating the sirtuin 1 (SIRT1)/NAD(P)H: quinone oxidoreductase 1 (NQO-1) axis. We found that NMN inhibited H2O2-induced cytotoxicity and senescence-associated protein expression, such as p16 and p21. Furthermore, NMN prevented H2O2-induced actin cytoskeletal disorganization via inhibiting reactive oxygen species (ROS) production. NMN increased NQO-1 mRNA and protein expression that in turn was abrogated by SIRT1 inhibition, suggesting that NMN-inducible NQO-1 was associated with SIRT1 activity. SIRT1 and NQO-1 inhibition attenuated the inhibitory effect of NMN on H2O2-inducible cytotoxicity, senescence-related protein upregulation, and actin cytoskeletal disorganization. Our findings provide new insights into the mechanism by which NMN exerts protective effects against vascular oxidative stress.
•NMN reduces H2O2-induced cytotoxicity and senescence-related protein upregulation.•NMN inhibits H2O2-induced actin cytoskeletal disorganization and oxidative stress.•NMN increases NQO-1 expression through SIRT1 activation.•NMN suppresses H2O2-induced damage in a SIRT1/NQO-1-dependent manner in HUVECs.
PDF
