Sex-specific metabolic and functional differences in human umbilical vein endothelial cells from twin pairs

Lorenz, Mario; Blaschke, Benjamin; Benn, Andreas; Hammer, Elke; Witt, E; Kirwan, Jennifer; Fritsche‐Guenther, Raphaela; Gloaguen, Yoann; Bartsch, Cornelia; Vietzke, Angelika; Kramer, Frederike; Kappert, Kai; Brunner, Patrizia; Nguyen, Hoang; Dreger, Henryk; Stangl, Karl; Knaus, Petra; Stangl, Verena

doi:10.1016/j.atherosclerosis.2019.10.007

Cited by 34 publications

(38 citation statements)

References 41 publications

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“…Recently, the sex-dependent expression of the miR548am-5p has been proposed to control the propensity of human male dermal broblasts to apoptosis through its ability to alter the relative expression of pro-and anti-apoptotic proteins [45], thus con rming the possible involvement of miRNAs in the regulation of sex-relative properties. In addition, the ability of female ECs to maintain higher levels of metabolites and a better energy balance under stressed conditions in comparison to male ECs has recently been described [18]. Accordingly, the secretome from stressed female ECs contains greater amounts of proteins involved in speci c metabolic pathways.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, HUVECs undergo in vitro angiogenesis, produce NO, and respond to shear stress, hyperglycemia, and in ammatory stimuli, thus recapitulating human disease pathophysiology. In addition, sex-dependent differences in gene expression and functional properties have been shown in male and female HUVECs by us and others groups [12][13][14]17,18,70], indicating HUVECs as a suitable model for studying cellular biological sex and increasing the translational value of basic research. Nevertheless, whether and through which mechanisms HUVEC dimorphic properties are lifelong conserved and regulated in adult male and female ECs still need to be deeper investigated.…”

Section: Limitations Of the Studymentioning

confidence: 98%

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Sex-dependent differences in the secretome of human endothelial cells

Cattaneo

Banfi

Brioschi

et al. 2020

Preprint

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Background: Cellular sex has been rarely considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and ‘omics approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms.Methods: We performed proteomic and gene ontology (GO) analyses of secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and western blot techniques, and efferocytosis through the ability of the macrophage cell line RAW-264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. Results: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison to female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggests that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seems to be independent on PTX3 expression. Conclusions: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

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Section: Discussionmentioning

confidence: 99%

Section: Limitations Of the Studymentioning

confidence: 98%

Sex-dependent differences in the secretome of human endothelial cells

Cattaneo

Banfi

Brioschi

et al. 2020

Preprint

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“…The HCT116 cell line was maintained in DMEM (Dulbecco's Modified Eagle Medium, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% or 20% FCS (Thermo Fischer Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Thermo Fischer Scientific, Waltham, MA, USA), 2 mM glutamine (Thermo Fischer Scientific, Waltham, MA, USA) and 1 g/L glucose (Sigma-Aldrich, St. Louis, MO, USA). HUVECs were isolated as previously described [17]. All cells were incubated in a humidified atmosphere of 5% CO 2 in air at 37 • C.…”

Section: Cell Culturementioning

confidence: 99%

“…Fetal calf serum (FCS) contains a large number of nutritional and macromolecular factors such as amino acids, sugars, lipids, and hormones essential for cell culture growth. For most cell lines, 10% FCS is used; however, some primary cells, such as human umbilical vein endothelial cells (HUVECs), need to be cultured in higher FCS conditions (e.g., medium with 20% FCS) [16][17][18][19][20][21][22]. To our knowledge, there have been no reports to date documenting the robustness and efficiency of harvesting and extraction protocols for metabolic profiling of adherent cells grown under high FCS conditions.…”

Section: Introductionmentioning

confidence: 99%

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions

et al. 2019

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A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials.

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“…Umbilical cord cells are subject to the effect of hormones produced by the fetal gonad in addition to maternal hormones. In fact, various studies have shown gender differences in male and female umbilical cords regarding gene expression, protein expression, cell viability after serum starvation, tube formation capacity, autophagy, intracellular ATP and metabolite levels, oxidative stress and angiogenesis [76][77][78][79]. Sexual dimorphism shown by HUVEC indicates that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVEC of both sexes should be used as a model to increase the quality and the translational value of research.…”

Section: Advantages and Disadvantages Of The Huvec Modelmentioning

confidence: 99%

Use of Human Umbilical Vein Endothelial Cells (HUVEC) as a Model to Study Cardiovascular Disease: A Review

et al. 2020

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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.

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Sex-specific metabolic and functional differences in human umbilical vein endothelial cells from twin pairs

Cited by 34 publications

References 41 publications

Sex-dependent differences in the secretome of human endothelial cells

Sex-dependent differences in the secretome of human endothelial cells

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions

Use of Human Umbilical Vein Endothelial Cells (HUVEC) as a Model to Study Cardiovascular Disease: A Review

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