Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors

William C

William C. decline in cardiac function (Fractional Shortening at day 30: 38.71 4.13% in GNF-6231 treated vs. 34.89 4.86% in vehicle-treated), prevented adverse cardiac remodeling, and reduced infarct size (9.07 3.98% vs. 17.18 4.97%). WNT inhibition augmented proliferation of interstitial cells, particularly in the distal myocardium, inhibited apoptosis of cardiomyocytes, and reduced myofibroblast proliferation in the peri-infarct region. studies showed that WNT inhibition increased proliferation of Sca1+ cardiac progenitors, improved survival of cardiomyocytes, and inhibited collagen I synthesis by cardiac myofibroblasts. Conclusion Systemic, temporary pharmacologic inhibition of the WNT pathway using an orally bioavailable drug immediately following MI resulted in improved function, reduced adverse remodeling and reduced infarct size in mice. Therapeutic WNT inhibition affected multiple aspects of infarct repair: it promoted proliferation of cardiac progenitors and other interstitial cells, inhibited myofibroblast proliferation, improved cardiomyocyte survival, and reduced collagen I gene expression by myofibroblasts. Our data point to a promising role for WNT inhibitory therapeutics as a new class of drugs to drive post-MI repair and prevent heart failure. Tyk2-IN-7 studies, GNF-6231 in DMSO as vehicle was used at a concentration of 100 nM; C-113 (vehicle: DMSO) was used at 1 M; and recombinant mouse WNT3A (vehicle: 01% BSA in PBS) was used at a concentration of 50 ng/mL after testing a range of concentrations between 25 ng/mlC100 ng/ml and demonstrating similar proliferative response (data not shown). Animals All procedures were carried out in accordance with Vanderbilt Institutional Animal Care and Use Committee (IACUC), and NIH guidelines. C57Bl/6J mice were purchased from the Jackson Tyk2-IN-7 Laboratory (Bar Harbor, ME) and maintained by PPY. TOPGAL [5] mice were a generous gift from Dr. Antonis Hatzopoulos (Department of Cell and Developmental Biology, Vanderbilt University). Cell lines Sca1+CD31?CD45? cells were isolated as previously described [25]. Briefly, H-2Kb-tsA58 transgenic mice in C57Bl/6 background expressing temperature sensitive thermolabile simian virus (SV40) large tumor (T) antigen under the ubiquitous mosue major histocompatibility complex (H-2Kb) promoterage 6- to 8-weekswere euthanized using overdose of Isoflurane followed by cervical dislocation. Hearts from five mice were dissected to isolate ventricular tissue, which was then minced and incubated with 10 ml of digestion solution (10 mg/ml collagenase II, 2.5 U/ml dispase II, 1 g/ml, DNase I, and 2.5 mM CaCl2) for 20 min at 37C. The non-myocytes were collected using Percoll gradient. A filtered myocyte-free single-cell suspension in PBS containing 0.5% BSA and 2 mM EDTA (PBS/BSA/EDTA) was treated with mouse BD Fc Block (clone 2.4G; BD Biosciences, San Jose, CA), and immune cells were magnetically removed with CD45 microbeads (Miltenyi Biotec Inc., Auburn, CA). After incubation with phycoerythrin (PE)-conjugated CD31 (clone 390; eBioscience, San Diego, CA) and fluorescein isothiocyanate (FITC)-conjugated Sca1 (clone E13-161.7; BD Biosciences) antibodies, CD31 positive cells were removed with anti-PE microbeads (Miltenyi Biotec). Tyk2-IN-7 Sca1+CD31? cells were magnetically isolated with anti-FITC microbeads (Miltenyi Biotec). Isolated Tyk2-IN-7 conditionally immortalized Sca-1+CD31?CD45? cells were plated at a density of 104 cell/cm2 and cultured on 1% gelatin-coated tissue culture dishes in DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin, and 2 mM glutamine and 10 ng/ml IFN- under a humidified atmosphere of air/CO2 (19:1) at 33C. Six days before experiments, cells were replated and cultured in the absence of IFN- at 37C. HL1 cell line, derived from mouse atrial cardiomyocytes was a kind gift from Dr. William C. Claycomb (Louisiana State University Medical Center, New Orleans, LA). These cells were cultured on gelatin/fibronectin (25 g fibronectin in 2 ml of 0.02% gelatin in water)- coated plates (fibronectin and gelatin from Sigma-Aldrich). The HL1 cell line was maintained at 37C in Claycomb medium (SAFC Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum (SAFC Biosciences), 100 M norepinephrine (Sigma) in 30 mM ascorbic acid (Sigma), 2 mM L-Glutamine (Sigma), penicillin, and streptomycin (Life Technologies, Grand Island, NY). iCell Plus Cardiomyocytes, which are primarily ventricular cardiomyocytes derived from human induced pluripotent stem (iPS) cells, were purchased from Cellular Dynamics International and maintained in 0.1% gelatin coated plastic plates in manufacturers proprietary maintenance medium. Primary mouse cardiac fibroblasts were isolated from the hearts of C57Bl/6 mice that were at least 12 weeks old following a previously Rabbit Polyclonal to PROC (L chain, Cleaved-Leu179) described protocol [26]. Briefly, mice were euthanized by overdose of isoflurane followed by cervical dislocation. Heart tissue was minced and placed into Kreba-Henseleit (Sigma; K3753a) buffer with 2.9 mM CaCl2 and 24 mM NaHCO3 containing a cocktail of.