Anti-TNF-α treatment modulates SASP and SASP-related microRNAs in endothelial cells and in circulating angiogenic cells

Abstract

Endothelial cell senescence is characterized by acquisition of senescence-associated secretory phenotype (SASP), able to promote inflammaging and cancer progression. Emerging evidence suggest that preventing SASP development could help to slow the rate of aging and the progression of age-related diseases, including cancer. Aim of this study was to evaluate whether and how adalimumab, a monoclonal antibody directed against tumor necrosis factor-α (TNF-α), a major SASP component, can prevent the SASP. A three-pronged approach has been adopted to assess the if adalimumab is able to: i) modulate a panel of classic and novel senescence- and SASP-associated markers (interleukin [IL]-6, senescence associated-β-galactosidase, p16/Ink4a, plasminogen activator inhibitor 1, endothelial nitric oxide synthase, miR-146a-5p/Irak1 and miR-126-3p/Spred1) in human umbilical vein endothelial cells (HUVECs); ii) reduce the paracrine effects of senescent HUVECs' secretome on MCF-7 breast cancer cells, through wound healing and mammosphere assay; and iii) exert significant decrease of miR-146a-5p and increase of miR-126-3p in circulating angiogenic cells (CACs) from psoriasis patients receiving adalimumab in monotherapy.TNF-α blockade associated with adalimumab induced significant reduction in released IL-6 and significant increase in eNOS and miR-126-3p expression levels in long-term HUVEC cultures.A significant reduction in miR-146a-5p expression levels both in long-term HUVEC cultures and in CACs isolated from psoriasis patients was also evident. Interestingly, conditioned medium from senescent HUVECs treated with adalimumab was less consistent than medium from untreated cells in inducing migration- and mammosphere- promoting effects on MCF-7 cells.Our findings suggest that adalimumab can induce epigenetic modifications in cells undergoing senescence, thus contributing to the attenuation of SASP tumor-promoting effects.

Keywords: Gerotarget; HUVEC; SASP; miR-126-3p; miR-146a-5p; replicative senescence.

Figure 1. Effect of TNF-α blockade on the expression of miRs and their target proteins in LPS-exposed THP-1 cells

A. TNF-α release into the culture medium by THP-1 cells after LPS treatment (1 μg/ml), expressed as pg/ml per 100,000 cells. B. MiR-146a-5p expression in THP-1 cells after 30 min or 5 h (hours) LPS exposure, with/without 24 h anti-TNF-α pretreatment, measured as fold change vs ctrl. C. MiR-126-3p expression in THP-1 cells after 30 min or 5 h LPS exposure, with/without 24 h anti-TNF-α pretreatment, measured as fold change vs ctrl. D. Irak1 and Spred1 expression levels and densitometry data in THP-1 cells after 30 min or 5 h LPS stimulation, with/without 24 h anti-TNF-α pretreatment. * Student's t test, p < 0.05. Data are mean ± S.D. of 3 independent experiments.

Figure 2. Effect of TNF-α blockade on the expression of miRs and their target proteins in senescent (SA-β-Gal > 50 %) and young (SA-β-Gal < 5 %) HUVECs with and without LPS-stimulation

A. TNF-α release into the culture medium by LPS-exposed (1 μg/ml) young and senescent HUVECs, expressed as pg/ml per 100,000 cells. B. MiR-146a-5p expression in young and senescent HUVECs with/without 24 h anti-TNF-α pretreatment, measured as relative expression (a.u). C. MiR-146a-5p expression in young and senescent HUVECs after 30 min or 5 h LPS stimulation, with/without 24 h anti-TNF-α pretreatment, measured as fold change vs ctrl. D. MiR-126-3p expression in young and senescent HUVECs with/without 24 h anti-TNF-α pretreatment, measured as relative expression (a.u). E. MiR-126-3p expression in young and senescent HUVECs after 30 min or 5 h LPS stimulation, with/without 24 h anti-TNF-α pretreatment, measured as fold change vs ctrl. F. Irak1 and Spred1 expression and densitometry data in young and senescent HUVECs after 30 min or 5 h LPS stimulation, with/without 24 h anti-TNF-α pretreatment. * Student's t test, p < 0.05. Data are mean ± S.D. of 3 independent experiments.

Figure 3. TNF-α blockade and HUVECs replicative senescence and SASP acquisition

A. Cumulative population doublings (CPDs) of HUVECs exposed to continuous anti-TNF-α treatment and of control cultures from 34 CPDs to complete growth arrest. Y axis, CPD; x axis, number of passages. B. Percentage of SA-β-Gal-positive cells at the beginning of the curve (young cells) and at its end (senescent cells), with/without anti-TNF-α treatment. C. p16/Ink4a mRNA expression in young and senescent cells with/without anti-TNF-α treatment. Data expressed as fold changes vs young cells. D. PAI1 mRNA expression in young and senescent cells with/without anti-TNF-α treatment. Data expressed as fold changes vs young cells. E. IL-6 release (pg/ml per 100,000 cells) into the culture medium by young and senescent cells with/without anti-TNF-α treatment. F. eNOS mRNA expression in young and senescent cells with/without anti-TNF-α treatment. Data expressed as fold changes vs young cells. G. MiR-146a-5p expression in young and senescent cells with/without anti-TNF-α treatment. Data expressed as fold changes vs young cells. H. MiR-126-3p expression in young and senescent cells with/without anti-TNF-α treatment. Data expressed as fold changes vs young cells. I. Irak1, Spred1 and IL1β expression and densitometry data (normalized to β-actin) in young and senescent cells with/without anti-TNF-α treatment. * Student's t test, p < 0.05. Data are mean ± S.D. of 3 independent experiments.

Figure 4. TNF-α blockade and bystander effect of HUVECs SASP

A. Drawing showing the experimental design. Conditioned medium from senescent cells (SA-β-Gal > 50 %) was mixed with 1/3 of fresh medium (with 30 % serum) and used to treat young cells (SA-β-Gal < 5 %) for 2 weeks, with/without anti-TNF-α treatment. Conditioned medium from young cells was used as control. B. Percentage of SA-β-Gal positive cells after 2 week exposure to conditioned media, with/without anti-TNF-α treatment. C. and D. eNOS and p16/Ink4a expression in cells treated with conditioned medium, with/without anti-TNF-α treatment. Data expressed as fold change vs control cells. * Student's t test, p < 0.05. Data are mean ± S.D. of 3 independent experiments.

Figure 5. Anti-TNF-α treatment effect on pro-motility activity and mammosphers (MS) promotion of HUVECs secretome on MCF-7 tumor cell

A. Drawing showing the experimental design. Senescent cells exposed to anti-TNF-α long-term treatment and senescent control cells were switched to fresh medium for 24 h and then their conditioned medium was mixed with 1/3 fresh DMEM. These mixtures, senescence conditioned medium (SEN-CM), and anti-TNF-α treated senescence conditioned medium (ATT SEN-CM) were used to treat MCF-7 in a wound healing assay. Conditioned medium from young cells was mixed with DMEM and used as control. B. Light microscopic photographs showing MCF-7 migration in the wound healing assay 0, 4 and 8 h after treatment with SEN-CM or ATT SEN-CM. C. Percentage of migrating cells after 4 and 8 h exposure to SEN-CM and ATT SEN-CM. Data expressed as percent of control. D. Representative pictures of MCF7-derived mammosphers (MS) promotion induced by conditioned media obtained from young cells (CTRL-CM), senescent cells (SEN-CM), and anti-TNF-α treated senescent cells (ATT SEN-CM). E. Quantification of MCF7-derived mammospheres in presence of different conditioned media. Data are presented as number of MS per well, at 3 and 7 days. Data are mean ± S.D. of 3 independent experiments. Scale bar 50 μm. * p < 0.05. * Student's t test, p < 0.05. Data are mean ± S.D. of 3 independent experiments.

Figure 6. In vivo anti-TNF-α treatment and endothelial senescence-associated miR expression levels in psoriatic patients

MiR-146a-5p A. and miR-126-3p B. expression in CACs from 13 psoriasis patients before and after 3 month anti-TNF-α monotherapy with adalimumab. P from Student's t test for paired samples.