Macrophage-based therapeutic approaches for cardiovascular diseases.

Despite the advances in treatment options, cardiovascular disease (CVDs) remains the leading cause of death over the world. Chronic inflammatory response and irreversible fibrosis are the main underlying pathophysiological causes of progression of CVDs. In recent decades, cardiac macrophages have been recognized as main regulatory players in the development of these complex pathophysiological conditions. Numerous approaches aimed at macrophages have been devised, leading to novel prospects for therapeutic interventions. Our review covers the advancements in macrophage-centric treatment plans for various pathologic conditions and examines the potential consequences and obstacles of employing macrophage-targeted techniques in cardiac diseases.

Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival.

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

Faster skin wound healing predicts survival after myocardial infarction.

Both skin wound healing and the cardiac response to myocardial infarction (MI) progress through similar pathways involving inflammation, resolution, tissue repair, and scar formation. Due to the similarities, we hypothesized that the healing response to skin wounding would predict future response to MI. Mice were given a 3-mm skin wound using a disposable biopsy punch and the skin wound was imaged daily until closure. The same set of animals was given MI by permanent coronary artery ligation 28 days later and followed for 7 days. Cardiac physiology was measured by echocardiography at baseline and MI days 3 and 7. Animals that survived until day 7 were grouped as survivors, and animals that died from MI were grouped as nonsurvivors. Survivors had faster skin wound healing than nonsurvivors. Faster skin wound healing predicted MI survival better than commonly used cardiac functional variables (e.g., infarct size, fractional shortening, and end diastolic dimension). N-glycoproteome profiling of MI day 3 plasma revealed α2-macroglobulin and ELL-associated factor 1 as strong predictors of future MI death and progression to heart failure. A second cohort of MI mice validated these findings. To investigate the clinical relevance of α2-macroglobulin, we mapped the plasma glycoproteome in patients with MI 48 h after admission and in healthy controls. In patients, α2-macroglobulin was increased 48 h after MI. Apolipoprotein D, another plasma glycoprotein, detrimentally regulated both skin and cardiac wound healing in male but not female mice by promoting inflammation. Our results reveal that the skin is a mirror to the heart and common pathways link wound healing across organs.NEW & NOTEWORTHY Faster skin wound healers had more efficient cardiac healing after myocardial infarction (MI). Two plasma proteins at D3 MI, EAF1 and A2M, predicted MI death in 66% of cases. ApoD regulated both skin and cardiac wound healing in male mice by promoting inflammation. The skin was a mirror to the heart and common pathways linked wound healing across organs.

Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology.

BACKGROUND: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. METHODS AND RESULTS: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. CONCLUSION: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Pleiotropic cardiac functions controlled by ischemia-induced lncRNA H19.

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation.

African-American Prostate Normal and Cancer Cells for Health Disparities Research.

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.

The Effects of a Mobile Wellness Intervention with Fitbit Use and Goal Setting for Workers.

Background and Introduction: There is strong evidence that worksite wellness programs can significantly improve the health profile of participating workers. To date, little is known about research on the effects of mobile wellness interventions in worksite settings. Furthermore, no studies have been conducted to evaluate mobile wellness interventions with activity trackers and tailoring strategies for physically inactive workers in manufacturing companies. This study aimed to examine the effects of a mobile wellness intervention with Fitbit and goal setting using brief counseling and text messaging among workers. Materials and Methods: A total of 79 (n = 79) workers from large manufacturing companies were allocated into an experimental group (n = 41) and a control group (n = 38). All participants were asked to wear an activity tracker (Fitbit Charger HR) during all waking hours for 5 weekdays. Participants in the experimental group received Fitbit, daily motivational text messaging, and biweekly counseling with a specifically designed workbook for 12 weeks, whereas Fitbit was only provided to the control group. Results: At the 12-week measurement, there were significant differences between the experimental group and control group on wellness (p < 0.001), physical activity behavior (p < 0.001), daily walking steps (p < 0.001), and physical activity self-efficacy (p < 0.001). Discussion and Conclusions: Although Fitbit facilitates an individual's activities by providing information about daily steps, the tracker itself, without additional goal-setting techniques, may be insufficient to encourage behavior change. These findings indicate that the mobile wellness intervention with Fitbit and goal setting using brief counseling and tailored text messaging is more effective for physically inactive workers.

Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing after myocardial infarction.

Matrix metalloproteinase (MMP)-9 increases in the myocardium with advanced age and after myocardial infarction (MI). Because young transgenic (TG) mice overexpressing human MMP-9 only in macrophages show better outcomes post-MI, whereas aged TG mice show a worse aging phenotype, we wanted to evaluate the effect of aging superimposed on MI to see if the detrimental effect of aging counteracted the benefits of macrophage MMP-9 overexpression. We used 17- to 28-mo-old male and female C57BL/6J wild-type (WT) and TG mice ( n = 10-21 mice/group) to evaluate the effects of aging superimposed on MI. Despite similar infarct areas and mortality rates at day 7 post-MI, aging TG mice showed improved diastolic properties and remodeling index compared with WT mice (both P < 0.05). Macrophage numbers were higher in TG than WT mice at days 0 and 7 post-MI, and the post-MI increase was due to elevated cluster of differentiation 18 protein levels (all P < 0.05). RNA sequencing analysis of cardiac macrophages isolated from day 7 post-MI infarcts identified 1,276 statistically different (all P < 0.05) genes (994 increased and 282 decreased in TG mice). Reduced expression of vascular endothelial growth factor A, platelet-derived growth factor subunit A, and transforming growth factor-ß3, along with elevated expression of tissue inhibitor of MMP-4, in macrophages revealed mechanisms of indirect downstream effects on fibroblasts and neovascularization. While collagen accumulation was enhanced in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both), the post-MI collagen cross-linking ratio was higher in WT mice ( P < 0.05), consistent with increased diastolic volumes. Vessel numbers [by Griffonia ( Bandeiraea) simplicifolia lectin I staining] were decreased in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both). In conclusion, macrophage-derived MMP-9 improved post-MI cardiac wound healing through direct and indirect mechanisms to improve diastolic physiology and remodeling. NEW & NOTEWORTHY Aging mice with macrophage overexpression of matrix metalloproteinase-9 have increased macrophage numbers 7 days after myocardial infarction, resulting in improved diastolic physiology and left ventricular remodeling through effects on cardiac wound healing.

Inflammatory cells and their non-coding RNAs as targets for treating myocardial infarction.

Myocardial infarction triggers infiltration of several types of immune cells that coordinate both innate and adaptive immune responses. These play a dual role in post-infarction cardiac remodeling by initiating and resolving inflammatory processes, which needs to occur in a timely and well-orchestrated way to ensure a reestablishment of normalized cardiac functions. Thus, therapeutic modulation of immune responses might have benefits for infarct patients. While such strategies have shown great potential in treating cancer, applications in the post-infarction context have been disappointing. One challenge has been the complexity and plasticity of immune cells and their functions in cardiac regulation and healing. The types appear in patterns that are temporally and spatially distinct, while influencing each other and the surrounding tissue. A comprehensive understanding of the immune cell repertoire and their regulatory functions following infarction is sorely needed. Processes of cardiac remodeling trigger additional genetic changes that may also play critical roles in the aftermath of cardiovascular disease. Some of these changes involve non-coding RNAs that play crucial roles in the regulation of immune cells and may, therefore, be of therapeutic interest. This review summarizes what is currently known about the functions of immune cells and non-coding RNAs during post-infarction wound healing. We address some of the challenges that remain and describe novel therapeutic approaches under development that are based on regulating immune responses through non-coding RNAs in the aftermath of the disease.

Proteomic analysis of the cardiac extracellular matrix: clinical research applications.

INTRODUCTION: The cardiac extracellular matrix (ECM) provides anatomical, biochemical, and physiological support to the left ventricle. ECM proteins are difficult to detect using unbiased proteomic approaches due to solubility issues and a relatively low abundance compared to cytoplasmic and mitochondrial proteins present in highly prevalent cardiomyocytes. Areas covered: Proteomic capabilities have dramatically improved over the past 20 years, due to enhanced sample preparation protocols and increased capabilities in mass spectrometry (MS), database searching, and bioinformatics analysis. This review summarizes technological advancements made in proteomic applications that make ECM proteomics highly feasible. Expert commentary: Proteomic analysis of the ECM provides an important contribution to our understanding of the molecular and cellular processes associated with cardiovascular disease. Using results generated from proteomics approaches in basic science applications and integrating proteomics templates into clinical research protocols will aid in efforts to personalize medicine.

A Mercaptoacetamide-Based Class II Histone Deacetylase Inhibitor Suppresses Cell Migration and Invasion in Monomorphic Malignant Human Glioma Cells by Inhibiting FAK/STAT3 Signaling.

Histone deacetylase inhibitors (HDACIs) have emerged as potential anticancer agents for the treatment of solid and hematopoietic cancers. Several HDACIs delay cell growth, induce differentiation, or activate apoptosis in multiple types of tumors, including glioblastomas. In the present study, we showed that the mercaptoacetamide-based HDACI W2 inhibits cell migration and invasion in monomorphic malignant human glioma cells. W2 treatment significantly decreased the activity and expression levels of matrix metalloprotease-2 in malignant A172 cells but not in U373MG cells. Key signaling pathways involved in cell migration and invasion, including PI3K-AKT, ERK-JNK-P38, and FAK/STAT3, were examined to identify the mechanism of action of W2. W2 increased the phosphorylation of AKT and altered cell migration and invasion in an AKT-independent manner. W2 inhibited the phosphorylation of FAK/STAT3, and treatment with a FAK/STAT3 inhibitor significantly suppressed cancer cell migration and MMP-2 activity in the presence of W2. In addition, W2 significantly inhibited the nuclear translocation of phospho-STAT3. Taken together, our results suggest that W2 suppresses cancer cell migration and invasion by inhibiting FAK/STAT3 signaling and STAT3 translocation to the nucleus in monomorphic malignant human glioma cells. J. Cell. Biochem. 118: 4672-4685, 2017. © 2017 Wiley Periodicals, Inc.

IL-10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation.

Inflammation resolution is important for scar formation following myocardial infarction (MI) and requires the coordinated actions of macrophages and fibroblasts. In this study, we hypothesized that exogenous interleukin-10 (IL-10), an anti-inflammatory cytokine, promotes post-MI repair through actions on these cardiac cell types. To test this hypothesis, C57BL/6J mice (male, 3- to 6-month old, n = 24/group) were treated with saline or IL-10 (50 µg/kg/day) by osmotic mini-pump infusion starting at day (d) 1 post-MI and sacrificed at d7 post-MI. IL-10 infusion doubled plasma IL-10 concentrations by d7 post-MI. Despite similar infarct areas and mortality rates, IL-10 treatment significantly decreased LV dilation (1.6-fold for end-systolic volume and 1.4-fold for end-diastolic volume) and improved ejection fraction 1.8-fold (both p < 0.05). IL-10 treatment attenuated inflammation at d7 post-MI, evidenced by decreased numbers of Mac-3-positive macrophages in the infarct (p < 0.05). LV macrophages isolated from d7 post-MI mice treated with IL-10 showed significantly elevated gene expression of M2 markers (Arg1, Ym1, and Tgfb1; all p < 0.05). We further performed RNA-seq analysis on post-MI cardiac macrophages and identified 410 significantly different genes (155 increased, 225 decreased by IL-10 treatment). By functional network analysis grouping, the majority of genes (133 out of 410) were part of the cellular assembly and repair functional group. Of these, hyaluronidase 3 (Hyal3) was the most important feature identified by p value. IL-10 treatment decreased Hyal3 by 28%, which reduced hyaluronan degradation and limited collagen deposition (all p < 0.05). In addition, in vivo IL-10 treatment increased fibroblast activation (proliferation, migration, and collagen production), an effect that was both directly and indirectly influenced by macrophage M2 polarization. Combined, our results indicate that in vivo infusion of IL-10 post-MI improves the LV microenvironment to dampen inflammation and facilitate cardiac wound healing.

Matrix metalloproteinases as input and output signals for post-myocardial infarction remodeling.

Despite current optimal therapeutic regimens, approximately one in four patients diagnosed with myocardial infarction (MI) will go on to develop congestive heart failure, and heart failure has a high five-year mortality rate of 50%. Elucidating mechanisms whereby heart failure develops post-MI, therefore, is highly needed. Matrix metalloproteinases (MMPs) are key enzymes involved in post-MI remodeling of the left ventricle (LV). While MMPs process cytokine and extracellular matrix (ECM) substrates to regulate the inflammatory and fibrotic components of the wound healing response to MI, MMPs also serve as upstream signaling initiators with direct actions on cell signaling cascades. In this review, we summarize the current literature regarding MMP roles in post-MI LV remodeling. We also identify the current knowledge gaps and provide templates for experiments to fill these gaps. A more complete understanding of MMP roles, particularly with regards to upstream signaling roles, may provide new strategies to limit adverse LV remodeling.

MMP-9 signaling in the left ventricle following myocardial infarction.

Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve both the stimulation of robust inflammation to clear necrotic myocytes and tissue debris and the induction of extracellular matrix (ECM) protein synthesis to generate an infarct scar. The collective changes in myocardial structure and function are termed LV remodeling, and matrix metalloproteinase-9 (MMP-9) is a key instigator of post-MI LV remodeling. Through direct molecular effects on ECM and inflammatory protein turnover as well as indirect effects on major cell types that coordinate cardiac wound healing, namely the infiltrating leukocytes and the cardiac fibroblasts, MMP-9 coordinates multiple aspects of LV remodeling. In this review, we will discuss recent research that has expanded our understanding of post-MI LV remodeling, including recent proteomic advances focused on the ECM compartment to provide novel functional and translational insights. This overview will summarize how our understanding of MMP-9 has evolved over the last decade and will provide insight into future directions that will drive our understanding of MMP-9-directed cardiac ECM turnover in the post-MI LV.

Defining the sham environment for post-myocardial infarction studies in mice.

The purpose of this study was to evaluate the effect of sham surgery in a minimally invasive surgical model of permanent coronary artery occlusion used to generate myocardial infarction (MI) in mice. Adult male C57BL/6J mice (3-6 mo old) were divided into five groups: day (D) 0 (no surgical operation), D1 Sham, D1 MI, D7 Sham, and D7 MI. A refined MI surgery technique was used to approach the coronary artery without the ribs being cut. Both sham and MI mice had the left ventricle (LV) exposed through a small incision. To test the effects of surgery alone, the suture was passed around the coronary artery but not ligated. The MI mice were subjected to permanent coronary artery ligation. The mice were killed at D1 or D7 postsurgical procedure. Compared with D0 no surgery controls, the D1 and D7 sham groups exhibited no surgical mortality and similar necropsy and echocardiographic variables. Surgery alone did not induce an inflammatory cell response, as evidenced by the lack of leukocyte infiltration in the sham groups. Analysis of 165 inflammatory cytokines and extracellular matrix factors in sham revealed that a minor gene response was initiated but not translated to protein levels. Collagen deposition did not occur in the absence of MI. In contrast, the D1 and D7 MI groups showed the expected robust inflammatory and scar formation responses. When a minimally invasive procedure to generate MI in mice was used, the D0 (no surgical operation) control was an adequate replacement for the use of sham surgery groups.

Radiotherapy-induced Immune Response Enhanced by Selective HDAC6 Inhibition.

Radiotherapy is a curative cancer treatment modality that imparts damage to cellular DNA, induces immunogenic cell death, and activates antitumor immunity. Despite the radiotherapy-induced direct antitumor effect seen within the treated volume, accumulating evidence indicates activation of innate antitumor immunity. Acute proinflammatory responses mediated by anticancer M1 macrophages are observed in the immediate aftermath following radiotherapy. However, after a few days, these M1 macrophages are converted to anti-inflammatory and pro-cancer M2 phenotype, leading to cancer resistance and underlying potential tumor relapse. Histone deacetylase 6 (HDAC6) plays a crucial role in regulating macrophage polarization and innate immune responses. Here, we report targeting HDAC6 function with a novel selective inhibitor (SP-2-225) as a potential therapeutic candidate for combination therapy with radiotherapy. This resulted in decreased tumor growth and enhanced M1/M2 ratio of infiltrating macrophages within tumors. These observations support the use of selective HDAC6 inhibitors to improve antitumor immune responses and prevent tumor relapse after radiotherapy.

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 3-O-ß-D-glucopyanosylspinasterol via blocking NF-κB and STAT1 signaling pathways in TNF-α and IFN-γ-induced HaCaT keratinocytes.

A phytosterol derivative, 3-O-ß-D-glucopyanosylspinasterol (spinasterol-Glc) isolated from leaves of Stewartia koreana was reported to inhibit LPS-induced cytokine production in macrophage cells. Thymus and activation regulated chemokine (TARC/CCL17) is produced in response to pro-inflammatory cytokines in keratinocytes, which is implicated in the development of inflammatory skin diseases. In present study, we investigated the effect of spinasterol-Glc on production of TARC/CCL17 induced by TNF-α and IFN-γ in human HaCaT keratinocytes. Spinasterol-Glc inhibited the mRNA and protein expression of TARC/CCL17 induced by TNF-α/IFN-γ in a dose-dependent manner. Inhibitors of c-Raf-1, p38 MAPK, and JAK2, suppressed the TNF-α/IFN-γ-induced production of TARC/CCL17, and phosphorylation of these signaling molecules were attenuated by spinasterol-Glc. The compound also inhibited phosphorylation of IKKα/ß and IκB-α, and reduced translocation of NF-κB to the nucleus. We demonstrated that spinasterol-Glc suppressed the NF-κB-driven and the GAS-driven expression of luciferase reporter gene induced by TNF-α and IFN-γ. In addition, spinasterol-Glc inhibited the DNA binding of NF-κB and STAT1 to its cognate binding site. These results suggest that spinasterol-Glc has effective inhibitory effects on production of TARC/CCL17 in keratinocytes via inhibition of NF-κB as well as STAT activation, and could be utilized for development of a potential therapeutic agent against skin inflammatory diseases.

Preclinical studies of YK-4-272, an inhibitor of class II histone deacetylases by disruption of nucleocytoplasmic shuttling.

PURPOSE: The HDAC shuttling inhibitor, YK-4-272 functions by restricting nuclear shuttling of Class II HDACs. Pre-clinical investigations of YK-4-272 bioavailability, pharmacokinetics, in vivo toxicity and tumor growth inhibition were performed to determine its potential as an HDAC shuttling disruptor for use in clinical applications. METHODS: The solubility, lipophilicity, in vitro metabolic stability, in vitro intestinal permeability, and in vivo pharmacokinetics of YK-4-272 were determined by HPLC methods. The anti-tumor activity of YK-4-272 was determined by monitoring athymic Balb/c nude mice bearing PC-3 xenografts. RESULTS: Oral bioavailability of YK-4-272 is supported by its solubility (0.537 mg/mL) and apparent partition coefficient of 2.0. The compound was chemically and metabolically stable and not a substrate for CYP450. In Caco-2 cell transport studies, YK-4-272 was highly permeable. The time-concentration profile of YK-4-272 in plasma resulted in a C ( max ) of 2.47 µg/mL at 0.25 h with a AUC of 3.304 µg × h/mL. Treatment of PC-3 tumor xenografts with YK-4-272 showed significant growth delay. CONCLUSIONS: YK-4-272 is stable and bio-available following oral administration. Growth inhibition of cancer cells and tumors was observed. These studies support advancing YK-4-272 for further evaluation as a novel HDAC shuttling inhibitor for use in cancer treatment.

Novel Paired Normal Prostate and Prostate Cancer Model Cell Systems Derived from African American Patients.

Prostate cancer is the most frequently diagnosed solid malignancy in men. African American (AA) men are at greater risk for developing prostate cancer, and experience higher mortality rates, as compared with Caucasian American men. However, mechanistic studies to understand this health disparity have been limited by the lack of relevant in vitro and in vivo models. There is an urgent need for preclinical cellular models to investigate molecular mechanisms underlying prostate cancer in AA men. We collected clinical specimens from radical prostatectomies of AA patients and established 10 paired tumor-derived and normal epithelial cell cultures from the same donors, which were further cultivated to extend the growth under "conditional reprogramming." Clinical and cellular annotations characterized these model cells as intermediate risk and predominantly diploid. Immunocytochemical analyses demonstrated variable expression levels of luminal (CK8) and basal (CK5, p63) markers in both normal and tumor cells. However, expression levels of TOPK, c-MYC, and N-MYC were markedly increased only in tumor cells. To determine cell utility for drug testing, we examined viability of cells following exposure to the antiandrogen (bicalutamide) and two PARP inhibitors (olaparib and niraparib) and observed decreased viability of tumor-derived cells as compared with viability of normal prostate-derived cells. Significance: Cells derived from prostatectomies of AA patients conferred a bimodal cellular phenotype, recapitulating clinical prostate cellular complexity in this model cell system. Comparisons of viability responses of tumor derived to normal epithelial cells offer the potential for screening therapeutic drugs. Therefore, these paired prostate epithelial cell cultures provide an in vitro model system suitable for studies of molecular mechanisms in health disparities.