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    Changes in CDKN2A-2B Expression Associate With T-Cell Phenotype... Translational Research 2019

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    Changes in CDKN2A/2B expression associate ® with T-cell phenotype modulation in ae atherosclerosis and type 2 diabetes mellitus ANGELA VINUE', SERGIO MARTINEZ-HERVAS', ANDREA HERRERO-CERVERA', VERONICA SANCHEZ-GARCIA, IRENE ANDRES-BLASCO, LAURA PIQUERAS, MARIA JESUS SANZ, JOSE TOMAS REAL, JUAN F. ASCASO, DEBORAH JANE BURKS, and HERMINIA GONZALEZ-NAVARRO VALENCIA, AND MADRID, SPAIN Previous studies indicate a role of CDKN2A/28/2BAS genes in atherosclerosis and type 2 diabetes melitus (T2DM). Progression of these diseases is accompanied by T-cell imbalance and chronic inflammation. Our main objective was to investigate @ potential association between CDKN2A/28/2BAS gene expression and T cell Phenotype in T2DM and coronary artery disease (CAD) in humans, and to explore: the therapeutic potential of these genes fo restore immune cell homeostasis and disease progression. Reduced mRNA levels of CDKN2A (p16), CDKN2B (p 15"), and CDKN2BAS were observed in human T2DM and T2DM-CAD subjects compared with controls. Protein levels of p16" and pisi"° were also dimin- ished in T2DM-CAD patients while CDK4 levels, the main target of p16" and p15™, were cugmented in T2DM and T2DM-CAD subjects. Both patent groups. displayed higher activated CD3+CD6%+ T cells and proatherogenic CD14++CD16+ monocytes, while CD4+CD25+CD127 regulatory T (reg cells) cells were decreased. Treatment of primary human lymphocytes with PD0332991, a pl6™#/ p15" mimetic drug and a proven CDKA inhibitor, increased Treg cells and the levels of activated transcription factor phosphoSTATS. In vivo PD0332991 treatment of atherosclerotic apoE-/— mice and insulin resistant apoE-/-Irs2+/— mice aug- mented Foxp3-expressing Treg cells and decreased lesion size. Thus, atherosclero- sis complications in 120M associate with altered immune cell homeostasis, diminished CDKN2A/2B/2BAS expression, and increased CDK4 levels. The present study also suggests that the treatment with drugs that mimic CDKN2A/2B genes could potential be considered as a promising therapy to delay atherosclerosis. ranstational Research 2019; 203:31- 48) “These authors comtibuted equally to this work From the Institute of Health Rescarch-INCLIVA, Valencia Spin: Endocrinology and Notion Department Hospital Clini Universitario, Depart: ‘ment of Medicine, University of Valencia, Valencia, Spain; CIBER de Diabetes y Enfermedaes Metabslcas (CIBERDEM),nsito de Sa Car Jos Il, Madrid, Spain: Deparment of Pharmacology, University of Valens, Valencia, Spain: Cento de Investigacion Prineipe Felipe (CIP), Valencia, Spain Subwitedfor Publication March 23,2018; rveived submitted August, 2018; accepted for publication August 7, 2018, Reprint requests: Herminia Gonzles-Navaro. Heath Research Insitute INCLIVA, AvdMenéndez Pelayo, 4, 46010 Valencia, Spain mal: herminiagonzalez@wv-s. 1931-S244/S- so from mater (© 2018 Elsevier Inc, All ights reserved ap: 10.1016. 2018.08.08, 3 ‘Abbreviations: BW = body woight; CAD = coronary artery dseaso: CDK Translational Research “January 2019 yoin-dependent inaulla resktonce; MAPK = mitogervactivated pxo- jgnal transducer ond activator of franscrip- tion: 720M = type 2 dlabetes melitus Th = Thelper cel Treg = regulatory Tell 32° VinuE et al finase: CVD = carcovaxoar dsoaeo I fein kinase Mets = metaboke synceome: STA ATA GLANCE COMMENTARY Angela V, etal Background Previous clinical and experimental studies indi- cate a protective role of CDKN2A/2B/2BAS genes in cardiovascular disease and type 2 diabetes mel- Titus (T2DM) which are characterized by immune cell imbalance. Translational significance {In the present study we observed that cardiovaseu- lar complications in T2DM in humans associate with diminished expression of CDKN2A/2B2BAS genes, reduced regulatory T (Treg) cells and increased proinflammatory monocytes. Interest- ingly, treatment of human lymphocytes with 'PD0332991, which is a proven CDK4 inhibitor and a drug that mimies CDKN2A/2B genes, aug- mented the levels of Treg cells. In addition, PDO332991 treatment of atherosclerotic mouse ‘models also enhanced Treg cells and diminished atherosclerosis. This study provides novel data to consider CDK4 inhibition via p15"™"/pi6™* mimetic drugs as a promising therapy for modu- lating atherosclerosis associated with T2DM. INTRODUCTION Cardiovascular disease (CVD) represents the first ‘cause of death worldwide! and is the main compli tion of type 2 diabetes mellitus (T2DM). Population aging and sedentary lifestyle patterns that promote obe- sity have produced a constant increase in the preva Tence of T2DM in the last decades.” Because of this, CVD will continue to be a major socioeconomic bur- den requiring novel therapeutic approaches. ‘T2DM is characterized by a chronic inflammatory state, which has been shown to accelerate the progression ‘of atherosclerosis, the most common cause of CVD. While initial stages of atherosclerosis are determined by ‘endothelial dysfunction, atheroma formation requires ‘monocyte activation and recruitment into the subendothe= lial space where this cell-type differentiates into macro- phages and generates vascular lesions by accumulating as foam cells.” The recruited immune T cells, although less abundant in lesions, have a key role in the coordination of the adaptive immune response and exert diverse effects ‘mediated by distinet T-cell subtypes." Proinflammatory T helper 1 (Thh1) cells are the most prevalent subtype and are proatherogenic, while Th2 cells are potentially anti- inflammatory but have a less-defined role in atherosclero- sis. Regulatory TT (Treg) cells are a minor anti-inflamma- tory subpopulation of CD4+ T cells with suppressive activity against CD4+ and CD8+ effector T cells and pro- mote ant-inflammatory macrophage phenotype:* Interest- ingly, an altered ratio between Treg cells and a Th subset associated with chronic inflammation, ThIT, also favors atherosclerosis progression, and decreased ‘Treg cells hhave been observed in human patients with vulnerable atheroma plaques and coronary artery disease (CAD).°° ‘The mechanism for these latter assocations is that the unbalanced interplay of the diferent T-cell subsets in pla- ques would lead to a defective resolution of the inflamma- tion and would generate obstructive vulnerable plaques, the hallmark of ischemic acute events.” Therefore, the fine-tuning of T cells to restore homeostasis has been cited as a promising treatment for vulnerable plaque and acute coronary syndromes.” ‘T2DM and CAD conditions have been associated with single nucleotide polymorphisms (SNPS) located i region of chromosome 9p21.3 near the Ink#/Arf loc ‘The InkArf locus encompasses the CDKN2A and CDKN2B genes, which encode the tumor suppressors plo, Altcmative reading frame (ARF) and p15", and CDKN2BAS gene, @ regulatory noneoxting antisense RNA named antisense noncoding RNA in the INK4 locus (ANRIL}” p16" and p15!" work as potent cell-eycle Inhibitors by binding to CDK4/6, and ARF exerts mainly proapoptotic activity.” Human and mouse studies have shown an atheroprotective effect of this region!" and others have shown CAD-associated SNPS affect Cik2a/ 2b/Anril mRNA levels and vascular cell growth." 1s0- forms of CDKN2BAS have been shown to regulate key lucose and fatty acid metabolism genes," while CDKN2B gene variants have been linked to reduced pan- creatic insulin secretion." Enhanced expression of the Inkd/Arf locus in mice alleviates insulin resistance (IR) associated with aging'” and diminishes hepatic steatosis by decreasing inflammation."* ‘Therefore, in the present study, we hypothesized that subjects exhibiting T2DM and CAD with altered T-cell homeostasis would exhibit decreased levels of CDKN2A/2B/2BAS genes. In addition, we wondered whether restoration of gene activity by mimicking its function could modulate T-cell homeostasis and would decrease disease progression, To answer these Tronsationol Research Velume 208; questions, we performed a cross-sectional study with subjects exhibiting T2DM and CAD and analyzed CDKN2A/2B/2BAS gene expression and leukocyte phe- notype. To evaluate the potential of CDKN2A/2B activ- ity as a therapeutic strategy, a mimetic drug of the protein gene products pi6"""" and pis, PDO332991, which is a selective inhibitor of CDK4, ‘was used in vitro experiments in isolated lymphocytes and in vitro studies employing mice prone to develop atherosclerosis and IR. MATERIALS AND METHODS Human subjects. The study was performed in acs dance with the ethical principles and guidelines for human research of the Helsinki Declaration, and was approved by the Hospital Clinico Universitario de Valencia ethical committee. All subjects gave wri ten informed consent. For the study, 120 unrelated individuals attending the outpatient clinic and selected over 18 months by the opportunistic sam- pling method were divided into controls, T2DM and ‘T2DM with CAD (T2DM-CAD). T2DM was dia; nosed when fasting plasma glucose was >126 mg/dL or when HbAIC was 26.5%, according to the American Diabetes Association criteria for T2DM."” Subjects with CAD were those with a history of myocardial infarction, angina, or evidence of signi cant ischemic disease as defined by the European Society Guidelines for CAD." Human carotid artery ultrasound evaluation for intima- ‘media thickness. A standardized imaging protocol was used for the intima-media thickness (IMT) measure ‘ments in agreement with the Mannheim consensus.” B-mode ultrasound imaging of the right and left carotid arteries was performed using a Siemens Sonoline G40 instrument equipped with 7—10 MHz broadband linear array transducers as previously deseribed.”! With the carotid dilatation and flow dividers as anatomic land- marks, the sonographer obtained high-resolution images of the common carotid (1. cm proximal to the bifurcation), the bifurcation (between dilatation and flow divider), and the internal carotid (1 cm distal to the flow divider). Carotid arteries and bifurcation were examined for the presence of atherosclerotic plaques defined as described.”! An experienced sonographer (S.M.-H.) performed all examinations. Intraobserver variability was examined in 20 subjects. The coef cient of variability of mean common carotid ii ‘media thickness (CC-IMT) was 5.2%. Human plasma parameters analysis and enzymé linked immunosorbent assay. Samples were collected after 12—14 hours fasting from an antecubital vein in Vinuéotal 33 tubes containing Ethylenediaminetetraacetic acid (EDTA) (BD Vacutainer) and centrifuged within 4 hours. Plasma was stored at 4°C and biochemical param- eters were measured by standard clinical methodology. Insulin was determined by radioimmunoassay. For ELISA, heparinized human whole blood (10 U hepainy mL) was obtained, plasma was isolated, and eytokines ‘were measured using the human IFNy, IL, and IL17A ELISA set (Diaclone, Besancon, France) and IL2 and ‘TGF DuoSet ELISA (RAD Systems, UK). Expetiments on isolated lymphocytes. Human periph- eral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy volunteers with Lympho- prep (Axis Shield PoC, Norway), plated in cel plates with 154% Fetal bovine serum (FBS)-RPMI1640+ cillin/Streptomycin/Amphotericin and m: humidified 5% CO atmosphere for 1 hour. After 1 hour, nonadherent cells which contained lymphocytes ‘were removed and plated. Lymphocytes were washed and plated in T-cell expansion media consisting of ‘TexMACS Medium, human T-cell TransAct (Milteny Biowech SL, Spain) and IL6, IL2, and TGFA (10 ng/ml each, PeproTech, London, UK) atthe suggested density (ypically at 1 x 10° cellvmL). After 7 days, Iymphocytes were treated with increasing doses (0, 5, 10, 50, and 100 nM) of PDO332991 (PDO332091 ise- thionate, PZ0199, Sigma) for 48 hours and then col- lected for flow cytometry and westem blot analy For insulin experiments, cells were plated, washed as before, and cultured in 15% FBS-RPMI1640-Penicil- Sureptomycin/Amphotericin media during 3 days with 400 nM of insulin or vehicle and then treated or not with PD0332991 at 50. aM for 24 hours and col- lected for flow cytometry of western blot analysi Mice, diets, ond reaiments. Animal care followed the ethical guidelines for animal research and was in accor- dance with the Directive 2010/6¥EU and with the approved protocols of the institutional committee of the University of Valeneia and INCLIVA. Male apok—/— (CSTBLI6I, Charles River) and apok-/ § _,COKNZA(pI6%) _COKN2A,pt4™) —_COKN2 coKNaeAs 3 aot toes pace bu fe kd ae hd 8 1 =" 04 a os z z 4 a 7 B i ae =e 2/4. ep it zd a oon a | ° D E ee Zoos Se | 208 s | 2 : 3 os 2 os 2 oa Boa! : B oz Boz a = "T2088 : "Da ‘0m a vt ————| H o $y? 340s ge 04 = ol ee eee) Cee) SAK oth ——————] tn es] tbulin| ——————| Fig 2. Expression analysis in PBMCs from control, T2DM, and T2DM-CAD human subjects. (A) mRNA expresion level of CDKN2A(p 16), CDKN2A(p1#™), CDKN2B, and CDKN2BAS normalized with the endogenous GAPDH mRNA levels ad eelaivized to contol group levels. 8) Proton expression of p16", pid and p18!" in PBMCs fom al 3 groups. Protein expression analysis of (C), CDKi (D), p27 and (H), 21 in PBMCs from the tee groups of subjects. Protein analysis of aetivated (F), 938 (phoxphetP3%p38) (G), ERK (pERKIERK) and (H), SAPKJNK {(®SAPK/pINK/SAPKIINK), Phosphorylated protin levels were normalized to al protein levels and unphosphorylated protein levels with tub Tin evo: Representative blots are son fr cach protein a-tbulin blots in panels Fare shown for lading contra. Staisticl analysis was r= formed using one-way ANOVA followed by Tukey's posthoc test. “Compared with contos. #Compaed with T2DM. CAD. coronary artery sisease; PBMCs, peripheral blood mononuclear cells Tronsationol Research Yeti 69 Vineet 3° g oe cosecoe0+ eozsvc012r-treq _cp2sscotz7» Fx 0.05 6 50) Es c i; 2 ne ea ———— rome 5 0 7 0 3 concer cou 3 3) 8 I Ed — 2 iad cs cos” co” co2s” eps” cos” cos” coas” CXCRS¢Th1 _ CORAVCCRE-Th2 CCRAFCCRS*THIT ' a + |30F * he ea 3 E 3 ~) Eh ——— D Bivehicie il pD0332991 50 nM 2 pro E 3 2 = z 2" ggrs Bis a a3 , #2 4c B41 Eos a qa & 3° g aaa 5 04 ago a Q ol o ron conn aos ee 2ctn [seen Fig & Effect of CDKS inhibition by PDO352991 in T cell sibpopoltions. (A) CD3+ and CDS+CD6% Tcl pereenages aftr tsatment ith easing doses (0S, 10, 30, snd 100 mM) of PDO32991 (PD). (B) Percentages of CD4¥CD2S+CDI2T Treg and CDISHCDI2I+ cells ated doses of PD asin A. Representative bots re shown at 0 aM PD conceatation for CD3, CDS, and CDI gating seatcgy below the bar grap. Representative plot of the pating strategy wsed for CDI27, CD25 are shown fr al concentrations of PD below the bar graphs (©) CDS 4CXCRSATHI, CDACCRISCCRS-TH2, and CDCCRSSCCR6ETHIT cell sbsts treated with inceaking doses of PD a in A. Representative plots ae shown at | aM PD concentration for CXCRS, CCR4, and CCR6 gating strategy below the har graphs. (D) Protcin expression analysis of PSTATSISTATS, pTyr70SSTATNSTATS, pSTATISTATI, pSer20XSMADS, and pSer24V/24SSMADS in Iymphocytes treated with 50M of 00332991. Phosphorylted proteins wore normalized to total profcin levels Representative los are sown foreach immunoblot and actin is shown as loading earl tat teal ays was ropeated moasires ANOVA analysis flowed Dunes mutple comparison test AC) and St dens test). "Compared with 0 aM Translational Research 40 VinuE otal January 2019 1 apo» vehicle Wi 2p0c + po0332901 ws Se mgrkghWiday nett A i B 7 age 60} pags 3 8 = en 3 2 3 Z 5 go d “ype Monoeyos Nowtophis cao | ce - tysom —Lysor j = veo, Ow eM || lon 4 = oe = c La ke" 3 g ae seid Es is § gal aay E 5 5 4 y eed i 3 Sane ie { “@ ie 7 rouse i cose Posten, | te 6 fey oi i 7 B Goaecoaseronpae ‘CoeecoasFoxpse E : on ma m7 Fd “ af Eu i K i te 5 3 imal Lgl] 3“ comexcns Coucenseone- Sowconcente F 4 EQ eom) ‘| rs 4 & Bron ad Fig 4, Effect of PD0832991 treatment in atherogenic dct-fed apoE~/~ mice. (A) Percentages of lymphocytes, monccytes, and neutrophils ama- lyzed in the CDAS+ leukocyte population PDO332991-and vehicle tated apoE~/~ mice. (B)Cirulting Ly6C*™- and Ly6C*-monceste subsets ienitied in the CDI15+CD4S+-monocyte population, (C) Ciruating CD34, CD3¥CDH%, CDE, CD4YCDHs, CDS, ad CDS+CDHH Tels Tronsationol Research Velume 208; insulin-stimulated lymphocytes (Fig $2, A P < 0.03 and P< 005) without affecting activated CD3+CD69+ T cells, PDO332991 treatment inereased the percentage of CD4+CD25+CD127— Treg cells in insulin-stimulated Iymphocytes (Fig $2, A, P = 0.01) but not in controls. Interestingly. CDK4 expression was increased in insul treated cells regardless of the PD0332991 treatment (Fig 2, B, P < 0202), which is consistent with the enhanced expression of this protein in T2DM subjects which are characterized by hyperinsulinemia, Thus, these data sug- gest that high insulin levels could enhance CDK4 expression, Given that decreased Treg cells associate with both high CDK4 levels and deranged T-cell popula tions, and that CDKG inhibition by PD0332991 restores ‘Treg levels, CDK4 could be, at least in part, responsible {or lymphocyte detrimental phenotype in IR conditions. Treatment with CDKA inhibitor PD0332991 modulates leukocyte phenotype in vivo. To evaluate the in vivo significance of CDK4 inhibition by PD0332991 in immune cell imbalance in the pathologic context of atherosclerosis, experiments were performed in mouse models. To evaluate the impact of PD0332991 on atherosel rosis, apoE—/— mice were treated with the inhibitor or vehicle S$ weeks and then characterized. Treatment with PDO332991 did not change BW, Total-, apoB-, HDL-Chotesterol, or triglycerides in apoE—-/— mice (Fig $3, A and B). Consistent with the above results, leukocyte analysis showed a decrease in lymphocyte percentage and number (Fig 4, A, P < 0.03; and Fig 83, C, p = 0.05) and an increase in the percentage but not in the absolute counts of neutrophils in PD0332991-treated apoF’-/— mice (Fig 4, A, P = 0.03; and Fig $3, ©). No differences were observed in the monocyte subsets (Fig 4, B) or in total CD3+, CD4 +, CD8+ or activated CD34CD69+, CD4+CD6%4, CD8+CD69+, T-lymphocytes between mouse groups (Fig. 4, C), Further analysis of CD4+ cells expressing Foxp3 showed enhanced levels of CD4+CD25+Foxp3 + Treg calls (Fig 4, D, P < 0.03) and no changes CD4 4CD25-Foxp3+ Treg subpopulation. No differences were found between mouse groups in the other CD4+ ‘Th subsets including CD44CXCR34ThI, CD44+CCRS 4+CCR6—Th2, and CD4+CCR4+CCRE+THIT (Fi 4, E), Similarly, no changes were observed in circulating IL2 or TGF cytokine levels between both groups of mice (Fig 4, P, Whole-mounted aorta analysis showed a decrease in lesion size in both the aortic arch and the thoracic aorta of PD0332991-treated apoE'-/— mice compared with Vinuéetot 41 vehicle-treated controls (Fig 5, A, P< 0.05 and P< 0.03). No differences were observed in the lesion size ‘measured in different regions of aortic cross-sections (Fig 5, B). To address whether PD0332991 treatment reduced pre-existing atheroma plaque _ lesions, PD0332991- and vehicle-treated apoE-/— mice were compared with apoE-/— mice-fed atherogenic diet during 3 weeks (Fig $4), Analysis showed enhanced lesion size in aortic arch and thoracie aorta in vehicle- uated mice compared with 3 weeks atherogenic diet- fed mice (Fig S4, A, P < 0.0001 and P < 0.001, respectively). In PD0332991-treated mice, lesions ‘were smaller than vehicle-treated apoE—/— mice (as in Fig, 5, A, P< 0.05) but in the aortic arch were signi cantly bigger than those in 3 weeks atherogenic dict- fed mice (Fig $4, A, P < 0.0001). Analysis in aortic cross-sections showed enhanced plaque size in vehicle- treated apoE—/— mice in the aortic root compared with 3 weeks atherogenic dicted mice (Fig $4, B, P< 0.01) without other changes in the other vascular regions analyzed. ‘Thus, PDO332991 treatment in ‘apoE-/— mice does not reduce pre-existing plaques but delays progression of atherosclerosis Further characterization of plaques showed no dif- ferences in the content of macrophages, VSMCs and T cells between PD0332991- and vehicle-treated apoE—/ — mice (Fig 5, C-B). PDO332991 treatment in apoE-/— mice did not affect collagen content in lesions but plaques had smaller necrotic core areas in PD0332991-treated_upok'—/— mice compared with controls (Fig 5, F, P < 0.0002 and P < 0.05). MMP analysis in plaques, which are involved in necrotic core formation in advanced plaques, showed enhanced MMP9 content (Fig 5, G, P < 0,004) and no changes in MMP? (Fig 5, G). Similarly, no differences were observed in Argl, iNOS, or Argl/iNOS ratio, which have been also associated to plaque stability (Fig 5, H), ‘To evaluate the impact of PD0332991 on atheroscle- tosis development in the context of T2DM, additional experiments were performed on apok'—/—Irs2+/— ‘mice. In addition of atherosclerosis, these mice develop characteristics of T2DM, such as IR and MetS. No changes were observed in BW or in the metabolic parameters analyzed including total- and HDL-choles- terol, triglycerides, fasting glucose, and glucose toler- ance between PD033299|-treated apok—/—Irs2+/— ‘mice and control mice (Fig $5, AD). Leukocyte anal- ysis revealed diminished percentage and absolute cell number of monocytes (Fig 6, A, P< 0.04 and Fig 55, E, P< 0.02) and lymphocyte absolute number < {Dy Circaing CD1CDISeFoxp and CDiCD25_FoxpieT call (E) Circulating CDICXCRIVTH, CDHCCRIFCCRE—TH2, and CDE “YCCR4SCCR5¥THIT cll sets in both groups. (F) HL2 and Te GE cytokine plasma levels in both groups of mice. Representative plos ofthe gat ing strategy of the Bow cytometry ae shown, Statistical analysis was performed using Stents test Translational Research 42 Vinuéotal “January 2019 D apoky-+ vehicle i apoE. + P00332991 Somg/KgBWxday m8 > wD AORTICARCH VEHICLE pD0332001 Fn "| ga | sole | § doa ae 3 3 ol z gn 2 iy 8 a : c D E Ea z z. 28d 5 3 ime Fides” 5S tel Me Peay ym ee #9 7 is * 4 asa 8 4 EI 3 UB: ai 8 - & z gl 5 Z i g : t Hamre i ie H coo Fig 5. Impact of PDOS32991 treatment in atherosclerosis atherosclerosis analysis in OM RO stained whole-mount anda the inti crosesections. atherogenic dit-fed apoE-/~ mice. (A) Bn face ot) Lesion size measured as the sea in rm? ‘Macrophage content (Mac area expressed ss Tronsationol Research Velume 208; (Fig 85, E, P< 0.01) in PDO332991-treated apoE—/—Irs2+/— mice compared with vehicle lreated mice. Further monocyte characterization showed enhanced Ly6C** monocyte subset and diminished Ly6C™ proinflammatory monocytes in PD0332991-treated apoE —/—Irs2+/— mice (Fig. 6, B, P < 0.05 and P< 0.05) compared with controls ‘T-lymphocyte analysis showed decreased CD3+ ‘Tolls (Fig 6, C, P=0.05) without changes in the a vated CD3+CD69+ T-cell subset (Fig 6, C). CD4+ ‘T-cell analysis showed enhanced CD4+CD25—Foxp3 + subpopulation in PD0332991-treated mice without changes in other Th subsets (Fig 6, D, P= 0.05). Circu- lating IL2_ and TGF cytokine levels remains unchanged between both groups of mice (Fig 6, E) Lesion analysis in the whole-mounted aorta showed decreased atheroma size only in the thoracic aorta of PD332991-treated apoE-/—Irs2+/— mice compared. with vehicle-treated controls (Fig 6, F, P < 0.02). No significant changes were observed in lesion in the aortic arch (Fig 6, F) or aort tions (Fig 6, G). Similarly, no changes were observed in the collagen content but necrotic core area was diminished in PDO332991-treated mice (Fig 6, H, P = 0.05). Analysis of MMP did not revealed differen- ces either in MMP9 or MMP2 between vehicle- and PD033299I-treated mice (Fig 6, 1). iNOS and Argl marker analysis revealed decreased iNOS plaque con- tent (Fig 6, J, P< 0.04) and an increased Argl/iNOS ratio in PD033299 I-treated mice compared with veh cle-treated controls (Fig 6, J, P < 0.03), DISCUSSION Co-existence of CVD and T2DM increases the risk of death in humans. ln this study, decreased expression of CDKN24/2B/2BAS mRNA and pi6!" and pis" proteins, which associate with CVD and T2DM,” was accompanied by diminished Treg cells and enhanced proatherogenic monocytes in T2DM and T2DM.CAD subjects. These patients also displayed reduced levels of the IL4 and IL2 anti-inflammatory cytokines that together with decreased Treg cells could indicate a reduced immune suppression capacity in these subjects. CDK4 protein levels, the main target of | < Vinuéotal 43 pl6™ and pis, were augmented in leukocytes from T2DM and T2DM-CAD humans. Treatment of human lymphocytes with a proven CDK¢ inhibitor, PD0332991, enhanced Treg cells in a dose-dependent ‘manner and increased the levels of activated STATS, a transcription factor involved in Treg cell differentia- tion. Moreover, PD0332991 treatment diminished ‘T-lymphocytes and enhanced CD4+Foxp3+-express- ing (Treg) cells in vivo in the atherosclerotic-prone apoE/— mouse model and in insulin-resistant apoE—/—Irs2+/— mice. Further analysis also showed reduced atheroma lesion formation in the whole aorta and reduced necrotic core area in plaques upon 'PDO332991 treatment in both mouse models although results were discrete in apoE -/—Irs2+/— mice. Given that CDK4 inhibition partly restored leukocyte homeo- stasis and prevented atheroma progression in these experimental models, this study suggests that treatment with CDKN2A2B mimetic drugs could be considered a therapeutic approach to delay atherosclerosis devel- ‘opment and stabilize plaques. Previous studies have shown a protective role of CDKN2A/2B/2BAS in chronic diseases including cancer, diabetes, and atherosclerotic disease. In mouse models of gain- and loss-of-function, aug- mented expression of CDKN2A/2B restores the glu- cose metabolism derangement associated with aging,'” inactivation of pl6"™"* or p19“ acceler- ates atherosclerosis|'* and Cdkn2a-deficiency pre~ disposes to thrombosis in hypercholesterolemic mice.” In the present study, T2DM and T2DM- CAD subjects which displayed enhanced atheroscle- rosis, as shown by the CC-IMT subrogate parame- ter, had reduced expression of CDKN2A (pI6'"**), CDKN2B, and CDKN2BAS mRNA levels. The pro- tein gene products p16" and pis"? were diminished in T2DM-CAD subjects which, in addi- tion to a previous acute ischemic event, displayed significantly augmented CC-IMT compared with controls and T2DM subjects. These results are con- sistent with human studies associating SNP variants of the CDKN2B and CDKN2BAS genes with changes in HOMA-IR™ and in fatty metabo- lism genes.'° Expression changes in CDKN2A/2B/ 2BAS are linked with CAD-associated SNPs'* and ‘with atherosclerosis risk SNPs.'' Altogether indicate ‘Perccnage and a absolute aca) (D), vascular smooth macle cel content (SMtaractin area expressed oper ‘contage anda bool area) and (E “THymphocjte mimber (CD3+ cells expres as nimber of cells per ara and as absolute number) measured in lesions of both groups of mice. (F) Neointimal collagen content and rccrotic core area (bah expressed as percentage and as shsoate area) analysis in Mins rome Goldner ‘sings. (G) MMP9 and MMP2 content in antic cros-ections. (HH) Ars and iNOS content (expressed as pr ‘and Arg iNOS ratio i artic cron sections from vehicle- and PDO332991 treated mice. Represent tive images ofthe different sainings ae shown, The discontinuous ines delineate the inns Statistical analysis ‘was performed using the Stott Translational Research 44 Vinuéotal “January 2019 Diapoe- irs2+/-+vehiclen=6 Ml spoE-/irs2+/- + PD0332991 50 mgiKgBWxday_ 155-6 A c Aa ry = = a oo vas | gf i zai ee 7 1 bags Bed) | 3 on : e ac oe a ac. 1 enact © Geta co cae D remserets E . ee A “ot a |4000} i fm a . soot i ao COO: cl: 5 dl scnareouccoaet caetcncan! cotace epcenes F pommeanct 5 G : ? j ir in: |: HE ule 15 8 we 5 ° + a) + Pe 2 i fils ei 2 y nore © ° ASCENDING AORTA 5 5 oor ‘AORTA i i venice ossscest H : souzz0et : SF a a as BS . 35 eI i 28 I J p03 i = oar Bes } 4 1 ° 5 VewicLe ‘po0732001 00352901 Fig 6, Impact of PDOS32991 treatment in atherogenic def apo’-/—Irs24/~ mice. (A) Pereentages of Iym- ‘hoeytes, monocytes, and neutrophils analyzed in the CD4S#-leukocyte population of PDO332991- and vehicle tweed apoB-/- mice. (B) Circulating Ly6C™- and Ly6C-monoeyte subsets identied in the CDLS| Tronsationol Research Velume 208; thatatheroselerosisin T2DMisaccompaniedbydecreased CDKN2A/2B2BASexpressionandupregulationofCDK4 aandithatcardiovascularcomplications,suchasCAD, prog- resswithdecreasedfunctionotp16"*"andp15"™** Thus, theseresultssuggestthatadefectivep16"*"andp1s™"- ‘mediatedinhibitionof theupregulatedCDK4couldhypo- thetically be responsible foracute CVD complicationsin T2DM. CDKN2A2B2BAS-CDK4 altered expression in PBMCs from T2DM and T2DM-CAD patients was accompanied by increased T-cell activation, decreased levels of Treg cells and enhanced proinflammatory CD14++CD16+ monocytes. Our results are consistent with several investigations in humans showing that complications of T2DM and atherosclerosis affect immune response. An imbalance in T-lymphocyte sub- populations is associated with atherosclerosis progres sion,” proinflammatory CD14++CDI6+ monocytes correlate with cardiovascular outcomes” and reduced circulating Treg number leads to plaque rupture.” The reduced anti-inflammatory IL4 and IL2 cytokine levels and the elevated proinflammatory monocytes seen in our study are also consistent with a low Treg number as they exert atheroprotective mechanisms through the induction of anti-inflammatory macrophages and pro- ‘mote anti-inflammatory eytokine seeretion.* ‘A shortcoming in our human in vivo studies is that ‘we cannot attribute CDKN2A/2B expression changes to specific immune cell population as expression studies were performed in whole PBMCs. Treatment of Iym- phocytes with PD0332991, which mimics CDKN2A/B function, increased ‘Treg. cells in a dose-dependent manner, while other Th subsets (Th17 or Thl) were affected only at cytostatic and anti-proliferative doses.” Moreover, when human lymphocytes. wer exposed (0 high insulin levels, a characteristic of IR subjects, CDK¢ protein levels were enhanced while ‘Treg cells were diminished. Treatment of these Iym- phocytes with PD0332091, restored Treg cell subpopu- lation, thus suggesting that CDK4 might potentially modulate T-cell homeostasis. Given that in vitro treat- ‘ment of lymphocytes with PD0332991 resulted in phe- notype modulation oT cells, changes in T-cell subsets « Vinuéotal 45 in vitro could be, at least in part, due to changes in CDKN2APB gene expression. Consistent with the in Vitro results, in vivo treatment of mouse models with suboptimal doses of PD0332991 elevated the levels of circulating CD4+Foxp3+ expressing Treg cells. These included CD4+CD25+Foxp3+ subset in atherosclerotic qpot—/— mice and CD4¥CD25—Foxp3+ cells in IR apok—/-Irs2+/— mice, which display suppressive functions." OF note, in apoE -/-Irs2+/~ mice, proin- flammatory Ly6C™ monocytes which promote ather- coma progression were decreased while anti- inflammatory Ly6C**-monocyte levels, a monocyte subtype involved in patrolling functions" were signifi- cantly augmented. Lesion size was modestly reduced but significantly in PD033209 treated apok—/— mice and IR mice. Consistent with a contribution of Treg cell modulation on atherosclerosis, previous stu demonstrated that ‘Treg cell expansion by different approaches diminishes atherosclerosis" while deple- tion of Treg cells promotes atheroma development in LDLrdeficient mice.” Although size reductions in atheroma plaques in our study could partially be atrib- ‘uted to changes in circulating immune Treg cells, given the ubiquitous expression of CDK4, the main target of PD0332991, other mechanisms might as well contrib- tute to delay atherosclerosis. Thus, CDK4 inhibition could limit cellular profiferation within the plaque, have effects in other atheroma cell types or even in dis- fant tissues that modulate atheroma growth. In this sense, previous investigations reported that CDK inhi- bition by PDO332991 reverses thrombosis induced by Westem-type diet feeding in LDLr-deficient mice,” by reducing platelet overproduction and activity, and diminishes hepatic steatosis induced by high fat feed- ing in WT mice by acting in the liver.” On the other hand, overexpression of CDKN2A/2B genes in mice, deereased systemic inflammation, total and activated T cells and proinflammatory Ly6C™-monocytes,"® while Cakn2a-ablation in the myeloid Tineage enhanced the percentage of Ly6C"-monocyte subset in mice.'” All these studies suggest that CDKN2A/2B gene modula- tion might potentially regulate inflammatory cell “+-monocye population. (C) Percentages of circulating CD3¥ and CD34CDO Tells. D) Percentage of Foups CDALCD2S4Foxpss, CD ¥CD2S-Foxps-expresing Treg eelh, CDACXCRITHI, CDCR 4CCR6~Th2, aed CDAFCCREFCCRE+THI7 cell suse in bath groups of mice. (B) TL2 and TGFB cytokine plasma Ives in hoth groups of mic. (F) Et fae aheroscleeos analysis in Oil Red-O sa ed whoe-mounted sort fom hoth groupe of mice.) Atherm lesion size measured a lesion area ands the tm mei aio inne crss-scctions.(H) Neointmal collagen content and necrotic core area analysis was performed in Mas som trichrome Goldner si ings. () MMPS and MMP2 content in aortic crose-oc ns from bath groups of mice. J) Are iNOS, and ArgI/INOS content i aot erosesections fom vehicle- an! PDUS3299 cated cw. Quanilicton in His expressed as bso sre in yma in Tan J ae expresed s percentage. Repre sentative images of the diferent sainings are shown, The discon 100 lines dinate the stina, Statistical alysis was performed using the Student's tes. MMP, matrix metallopscinases 48 Vinugotal phenotype in diverse experimental settings and this could be, at least in part, responsibly of disease regres- ‘One consistent observation in our study is that mice treated with PD0332991 along with increased Treg cells exhibited diminished necrotic core area, indicat- ing improved plaque stability. Interestingly, other investigations have shown that treatment of athero- sclerotic mouse models with Tregs improves sta zation of advanced atherosclerotic lesions.”° Several potential mechanisms associated with Treg cell action have been described for improvement of pla- {que stability. T cells might secrete’ or influence macrophages to secrete” MMPs which are major modulators of plaque vulnerability.” In our study, Treg cell increase and reduced necrotic core in apoE/— mice coincided with enhanced MMP9 content which has been reported to promote stable plaque phenotype." In addition, Treg cells also con- tribute to lesion stability by inducing proresolving and anti-inflammatory M2. phenotype in macro- phages” which are characterized by a higher Arg ‘expression and a low content of iNOS, an MI proin- flammatory marker. Irs2+/—apoE-/— mouse plaques displayed increased ArgI/NOS ratio, suggesting a potential prevalence of M2 macrophages in lesions which is in agreement with improved plaque stability and with a prevalence in blood of Ly6C** mono- cytes in this mouse model. These latter results are in the line of studies describing the implication of at nase I in plaque stability through diminished inflam- mation’? and of previous results. by us associating M2 macrophage polarization and plaque stability in Irs2+/—apoE-/— mice.”” All together suggest that PDO332991 treatment increases Treg cells in mouse ‘models and that these cells could potentially improve plaque stability through differem mechanisms depending on the mouse model employed. One imitation of the study is that, as expected, T2DM and T2DM-CAD subjects had a higher fre- ‘quency of oral hypoglycemic and lowering lipid drug medication use than controls. In addition to its primary effects, the most commonly used treatments, statins, and metformin, exert beneficial anti-inflammatory cffects through diverse mechanisms including modula- tion of Treg cell biology and monocyte arrest." However, the medication did not appear to interfere with the study as T2DM and T2DM-CAD exhibited decreased Tregs and enhanced inflammatory: mono- cytes and lymphocyte activation, OF note, white blood cell count has been shown to correlate with risk for developing T2DM,"* but no differences were observed in the leukocyte count among our cohorts of patients Translational Research “January 2019 which could be probably due to the mentioned drug treatments CD4+ T cell lineage commitment is specified by eytokine environment through Janus kinase/STAT sig- naling and activation of specific transcription factors."° ‘Thus, activation of STATI by IFNy promotes Th phe~ notype, TGF- and ILG/STATS-signaling mediate ‘Thi? differentiation and TGFA- and IL-2/STATS-sig- naling increases Foxp3 expression for Treg differentia~ tion.” In our study, treatment with PD0332991 elevated activated STATS in human lymphocytes while, consistent withthe absence of effect on THI or Thl levels at 50 nM, no changes were observed in either STATI or STAT3 transcription factors. These results might indicate a potential role of CDK4 in mod- ulating STATS protein activation, In fact, noncanonical functions of cyclin-dependent kinases include activat ing and repressing transcription factors essential for T- cell differentiation by phosphorylation. Thus, cyclin E-CDK2 complex negatively regulates Foxp3 by phosphorylation, CDK6 activates STAT3,"* and CDK4 and CDK2 can repress the activity of SMAD3,"” which has been shown to modulate ThI7 and Treg differentia- tion mediated by TGF9.”” However, no changes in the active (pSer423/425) or inactive (pSer208) phosphory- lated status of SMAD3 were observed upon CDK4 Inhibition by PD0332991. Whether CDK4 activity or CDKN2A/2B genes by inhibiting this kinase play a role in lymphocyte phenotype through interaction with key transcription factors for T cell lineage commitment cannot be drawn from our study. Therefore, further experimental data are needed to precisely define the role of CDK4 in T-cell homeostasis and phenotype commitment In summary, our observations indicate that progres- sion of atherosclerosis in T2DM and acute cardiovas- cular complications in humans associated with decreased CDKN2A/2B2BAS expression, enhanced CDK¢ levels, and immune cell imbalance (diminished ‘Treg cells, augmented T-cell activation, and elevated proatherogenic monocytes). In_ addition, treatment with PDO332991, a pi6"™/pis™" mimetic drug and a proven selective CDK4 inhibitor, increased Treg levels in vitro in human lymphocytes and in ivo in animal models of atherosclerosis and IR which could have an impact on atheroma develop- ment. Therefore, the present study provides novel human and experimental data to consider CDK4 inhibition as a promising therapy to delay athero- sclerosis acceleration associated with T2DM. Fur- ther experiments in animal models are needed to fully assess whether CDK4 inhibition might be a suitable therapy to explore. Tronsationol Research ‘Volurne 203 ACKNOWLEDGMENTS We thank A. Viguer for help with human studies, G. Herrera for flow cytometry assistance, and Dr A. Diaz for help in animal studies. AU the authors have read the authorship agreement of the journal. Conflicts of Interest: S.M-H., A.V., and H.G-N. are inventors. on a patent application submitted by INCLIVA that covers the PD0332991 results described in this paper. The rest of the authors have read the jour- nal’s policy on disclosure of potential conflicts of inter- ‘est and have declared no conflict of interest. Author Contributions: S.M.-H. and A.V. participated in the study design, the acquisition and interpretation of all data (clinical and experimental), and in writing the manuscript. V.S.-G, performed cytometry and lym- phocyte experiments, LAB. and A.H.-C. performed the characterization of lesions in the mouse model. LP. and MS. helped in the design of the inflamma- tory characterization experiments and critically revised the manuscript, JF.A. and JT.R. helped in the selec- tion of patients and in the clinical studies. D.J.B. parti ‘pated in the study design and critically revised the manuscript, H.G.-N, conceived and designed the study, participated in and supervised the acquisition and inter- pretation of the data and wrote the manuseript. All the authors have read the joumal's authorship agreement and the manuscript has been reviewed by and approved by all named authors. Funding: This study was supported by grants from the Carlos III Health Institute (PI13/00834 and PLIG/ (00091) and the European Regional Development Fund, and CIBERDEM. H.G.-N. is an investigator from the “Miguel Servet" programme (CP16/00013). 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