Why charcoal stripped serum

In D , ATP luminescence was normalized to the corresponding protein levels. F- and M-ECs are orange and blue, respectively. Cell number was expressed as percent of control, i. Solid bars: SM replaced with SM. We have previously shown that, besides cell growth, CSS-medium significantly inhibited the ability of ECs to sprout from spheroids embedded in a 3-D collagen gel to study in vitro angiogenesis [ 7 ]. In this assay, spheroids maintained their ability to sprout when cultured in Standard medium Fig 3A , left panels whereas the outgrowth of capillary-like structures was significantly reduced in spheroids incubated in CSS-medium Fig 3A , right panels.

Quantification of the number of sprouts and of their cumulative and average length showed that: i the basal capillary outgrowth was super imposable between F- and M-EC spheroids incubated in Standard medium; ii all the processes were severely inhibited in the presence of CSS-medium in ECs of both sexes Fig 3B, 3C and 3D. However, we observed that the CSS-medium was more effective in impairing in vitro angiogenesis in M- compared to F-ECs for all the parameters tested.

Photographs were taken 24 h later. Estrogens are important regulators of eNOS expression and activity through both genomic and non-genomic mechanisms [ 16 ]. Nevertheless, the female eNOS expression remained higher by about 1. In addition to hormones and peptides, charcoal stripping deprives serum of fatty acids FAs [ 17 , 18 ].

Recently, Schoors et al. We therefore investigated whether the loss of FAs might be responsible for the CSS-induced cell growth inhibition. Palmitic acid was chosen since it is the most common saturated FA in human plasma. In addition, its metabolic fate in ECs has been characterized [ 19 ].

Data are expressed as percent of control, i. Overall, our data indicate that the EC impairment in CSS-medium was primarily due to the absence of essential metabolic components e. Nevertheless, when we added palmitic acid or acetate to the CSS-medium, we did not observed a full rescue Figs 5 and 6. We thus tested whether E2 or DHT supplementation might further restore the proliferative capability of ECs cultured in these partially reconstituted media.

The presence of E2 or DHT did not significantly affect the results. The presence of E2 did not significantly affect the results. We observed that E2 supplementation 10 nM was per se ineffective in preventing the lack of sprouting in ECs of both sexes Fig 7C. Epidemiological and pharmacological studies have shown that estrogens exert a protective effect on vascular endothelium, resulting in a different prevalence and severity of CVDs in female and male population [ 1 , 2 ]. In humans, both male and female ECs express sex hormone receptors [ 4 , 21 ] but in vitro effects of E2 and DHT have been mainly described in ECs not segregated for sex.

To evaluate sex hormone activities in vitro , nominally hormone-free media- where fetal bovine serum is substituted with CSS in phenol red-free media—are commonly used. In our experiments, however, the failure in endocrine responses was not restricted to sex hormones. CSS lacks other hormones relevant for EC function, including thyroid hormone [ 11 , 18 ]. Despite physiological concentrations of T3 [ 22 ] are able to induce in vitro and in vivo angiogenesis [ 23 , 24 ], our results indicate that thyroid hormone was unable to restore both female and male EC growth when added to CSS-medium.

However, in some of these studies, hormone starvation was maintained for few hours [ 25 — 27 ] and media different from were used [ 21 , 27 — 29 ]. Moreover, ECs from non-human species e. Therefore, we hypothesize that some other components rather than hormones essential for the maintenance of EC properties are lost in the CSS. A potentially rather important failure in CSS may be represented by lipids that are removed from serum by charcoal stripping [ 11 , 17 , 18 , 32 ].

Thus, FAs are crucial components of serum lost in CSS and required for the maintenance of EC growth and in vitro morphogenetic properties. As a matter of fact, it has been demonstrated that the pharmacological or genetic block of FAO cause growth and sprouting inhibition in ECs as the result of an inadequate synthesis of nucleotides critically required for DNA replication [ 19 ]. The addition of FAs or acetate, by restoring mitochondrial FAO, provides carbons for the tricarboxylic acid TCA -cycle-derived amino acids for instance, aspartate, the precursor for the de novo nucleotide synthesis , and rescues EC proliferation and in vitro angiogenesis [ 19 ].

These results suggest that the loss of other lipidic serum components endowed with biological activity toward ECs, such as active sphingolipids [ 32 , 33 ], might contribute to the CSS-induced inhibitory effects. Lack of metabolites was however totally overcome by the addition of standard medium that fully restored EC metabolic activity.

This result is in accordance with our recent data showing that male ECs strictly rely on proliferation for in vitro sprouting at variance with female ECs that rather depend on migration [ 6 ]. Interestingly, male EC sprouting was less efficiently counteracted by the addition of acetate in comparison to female ECs.

These results suggest that still unknown differences might exist in metabolic pathways between female and male ECs. As a matter of fact, diverse serum metabolite profiles have been shown in men and women, and sex-specific differences commonly influence whole metabolic pathways rather than randomly affect distinct metabolites [ 34 ].

Remarkably, FAs, sphingolipids, carnitine and acetyl-carnitine are among the metabolites that display sex-differential concentration patterns [ 34 ]. Experiments are ongoing in our laboratory to elucidate potential metabolic differences between male and female ECs. Nevertheless, female eNOS expression remains higher by about 1. Further experiments will be performed to establish whether the female decrease in CSS, although not significant, might reveal an estrogen-dependent component that contribute to the higher constitutive female eNOS expression.

Collectively, our experiments indicate that human primary macro-vascular ECs cultured in CSS-medium experienced metabolite deprivation, and this withdrawal greatly affected their endocrine response phenotype.

Find more information on the Altmetric Attention Score and how the score is calculated. Charcoal-stripped fetal bovine serum CS-FBS is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer CaP cells. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.

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Otsuka T. Information updated. Close Confirm. GTX Reference 2. Datasheet File. See all Fetal Bovine Serum products. Datasheet Reference. Summary FBS-charcoal stripped Triple 0. This treatment minimized variability in sera lot to lot and normally improves immunoassay systems.