Additional file 1 of Weight-dependent and weight-independent effects of dulaglutide on blood pressure in patients with type 2 diabetes

Additional file 1. Supplemental Methods and Tables S1-S9

Mycophenolic acid induces senescence of vascular precursor cells

<div><p>Objective</p><p>Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function.</p><p>Methods</p><p>We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects.</p><p>Results</p><p>Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated β-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA.</p><p>Conclusion</p><p>Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells <i>in vitro</i>, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies <i>in vivo</i> to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.</p></div

MPA inhibits cell proliferation in a dose dependent manner.

<p>(A) Cell growth curve was evaluated using trypan blue staining to measure viable cells at 24, 48 and 72 hours. 1–5 μM concentration of MPA inhibited cell proliferation (n = 3). (B) Percentage of single cord blood-derived ECFC undergoing at least one cell division 14 days after MPA treatment (n = 3). (C) Percentage of cell clusters, LPP-ECFCs and HPP-ECFC 14 days after MPA treatment using single cell analysis (n = 3). (D) Proliferation of ECFC in the absence or presence of MPA (0.1, 0.5 and 1 μM) measured after 1, 3, 5 and 7 days by a FACS-based CFSE dilution assay. While 0.1 μM MPA did not impact ECFC division, MPA at higher concentrations significantly diminished ECFC division (n = 4). Results represent the mean ± SD. *P <0.05, **P<0.005, *** P<0.001 compared to vehicle. Abbreviations: ECFC = endothelial colony forming cell, EGM = endothelial growth medium, MPA = mycophenolic acid, LPP-ECFC = low proliferative potential—endothelial colony forming cells, HPP-ECFC = high proliferative potential-endothelial colony forming cells, CFSE = carboxyfluorescein succinimidyl ester.</p

ECFC identification criteria.

<p>ECFC display clonogenic potential (A), cobblestone morphology [scale bar = 100 μm] (B) and have ability to form cord-like structures in 2D Matrigel dishes (C) and vessel-like structures in 3D collagen matrix (D) in vitro. Abbreviations: ECFC = endothelial colony forming cell, MPA = mycophenolic acid, LPP-ECFC = low proliferative potential—endothelial colony forming cells, HPP-ECFC = high proliferative potential-endothelial colony forming cells.</p

Guanosine reversed MPA’s inhibitory effect on cell proliferation and senescence markers.

<p>Cell proliferation using trypan blue exclusion assay shows that guanosine is not cytostatic or inhibitory (A), and that addition of guanosine rescues ECFC proliferation in a dose dependent manner (B). Relative fold expression of senescence associated gene markers p16^INK4a, p21^WAF and p27^KiP1(C). Phosphorylation of p53 normalized to total p53 level in cell lysates (D). Results represent the mean ± SD compared (n = 3). *P < 0.05, ***P <0.001 relative to vehicle. Abbreviation: MPA = mycophenolic acid, ECFC = endothelial colony forming cell, OD = optical density, ph-p53 = phosphorylated p53, tot-p53 = total p53.</p

MPA treatment impairs ECFC migration.

<p> Cell migration was assessed using an <i>in vitro</i> scratch assay with and without MPA treatment. Upper panel: Representative phase-contrast images of ECFC incubated with vehicle control media at time 0 hour after wounding (A) and closure of the gap after 15 hours (B). ECFC incubated with 1 μM MPA at time 0 hour after wounding (C) and presence of a gap after 15 hours (D). Lower panel: Quantification of gap distance after 15 hours of incubation. Gap fully closed in vehicle treated wells while a gap is still clearly visible in cells treated with 1 μM MPA (25.8% ± 10%, *p<0.005) compared to vehicle (n = 4 to 6) Abbreviations: MPA = mycophenolic acid, ECFC = endothelial colony forming cells.</p

Diminished ECFC vasculogenic function after MPA treatment.

<p>Left panel shows representative photomicrographs (magnification, 5x) of TdTomato ECFC following no treatment (vehicle) or treatment with 2.5 μM MPA in 2D assay (A) and 3D assay (D). 2D Matrigel assay showed that there is decreased average total cord length (C) and cord area (B) after MPA treatment. 3D collagen assay showed decreased total vessel area (E) after MPA treatment. *P <0.05, **P <0.005 compared to vehicle (n = 3). Abbreviations: ECFC = endothelial colony forming cells, 2D = two-dimensional, 3D = three-dimensional, EGM = endothelial growth medium, MPA = mycophenolic acid, mm = millimeter.</p

MPA induced ECFC senescence.

<p>MPA treated cells have increased SA-β-gal staining and higher senescence related p21^WAF gene expression. (A) Photomicrograph of SA-β-gal staining of ECFC cultured with vehicle and 1 μM for 5 days. Blue color (arrow) indicates cellular senescence. (B) Quantification of SA-β-gal staining. At least 500 cells per individual sample were counted. The number of SA-β-Gal-positive cells treated with 0.5 and 1μM MPA concentrations were significantly greater than untreated cells. (C) Gene expression of p16^INK4A, p21^WAF and p27^Kip1 in untreated and treated ECFC normalized to B2M products. Results represent the mean ± SD compared (n = 3). *P < 0.05, **P <0.001 relative to vehicle control. Abbreviations: MPA = mycophenolic acid, SA-β-gal = senescence associated-beta galactosidase, ECFC = endothelial colony forming cell, B2M = beta-2-microglobulin.</p