Mitochondria regulate proliferation in adult cardiac myocytes.

Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.

Epithelial Squamous Metaplasia and Dysplasia in Inflammatory Nasal Polyps: An Observational Study.

Nasal polyposis (NP) is characterized by polypoid outgrowths of chronically inflamed respiratory mucosa. The presence of squamous metaplasia and dysplasia on the mucosal surface of nasal polyps (NPs) represents different manifestations of epithelial atypia. The aim of this investigation was to evaluate the presence of epithelial squamous metaplasia and dysplasia in ethmoidal NPs. This retrospective analysis of prospectively collected data involved 212 patients with NP undergoing endoscopic ethmoidectomy. To evaluate possible etiological factors for epithelial atypia, the patients in whom we histopathologically detected the presence of epithelial atypia were compared with patients with "normal" NPs in accordance with the following characteristics as found in the patients' medical records: gender, age, main symptoms, preoperative extent of sinus disease on computed tomography, atopic status, aspirin sensitivity, cigarette smoking, and occupational exposure to different noxious factors. Epithelial atypia were detected histopathologically in 44 (20.7%) NP patients, whereas features of "true" dysplasia were found in only 1 (0.5%) patient. The presence of atypia was more frequent in males than in females (P = .008). The association with aspirin-exacerbated respiratory disease and with long-term occupational exposure to different noxious chemicals, especially in workers exposed to salts of heavy metals, was more frequent in NP patients with epithelial atypia than in patients without atypia (P = .023; P = .006, respectively). Our results suggest epithelial atypia in NPs are associated with aspirin sensitivity and occupational exposure to different noxious chemicals. Although extremely rare, epithelial dysplasia may occasionally be noted in NPs, a fact potentially useful for both rhinologists and pathologists.

Patient and provider factors associated with the noninitiation of tamoxifen for young women at high-risk for the development of breast cancer.

We sought to identify factors associated with disparities in tamoxifen utilization among young patients at high-risk for developing breast cancer. We identified 67 premenopausal, high-risk women age 35-45, without surgical prophylaxis, who did not initiate tamoxifen. Factors associated with noninitiation were examined. About 37% of patients had no documented provider-based discussion regarding initiation. Type of high-risk diagnosis was the only factor associated with a provider-based discussion (P = .03). For patients offered tamoxifen, primary reasons for noninitiation were perceived minimal benefit (66.7%), fertility concerns (16.7%), and concerns about side effects (7.1%). Implementation of comprehensive educational strategies regarding the benefits of tamoxifen should be facilitated to improve initiation among young high-risk patients.

Synergistic effect of eribulin and CDK inhibition for the treatment of triple negative breast cancer.

Activation of CDK2 in triple negative breast cancer (TNBC) can contribute to non-canonical phosphorylation of a TGFß signaling component, Smad3, promoting cell proliferation and migration. Inhibition of CDK2 was shown to decrease breast cancer oncogenesis. Eribulin chemotherapy was used effectively in the treatment of TNBC. To this end, we tested therapeutic efficacy of a novel CDK2/9 inhibitor, CYC065, eribulin, and the combination of CYC065 and eribulin in 3 different TNBC cell lines, and an in vivo xenograft model. Specifically, we characterized cell proliferation, apoptosis, migration, cell cycle associated protein expression, treatment-related transcription factor activity, and tumor growth in TNBC. Treatment with CYC065 and eribulin in combination had a superior effect on decreasing cell proliferation, inducing apoptosis, and inhibiting migration in TNBC cell lines in vitro. Combination therapy inhibited non-canonical Smad3 phosphorylation at the T179 site in the protein linker region, and resulted in increased p15 and decreased c-myc expression. In a transcription factor array, combination treatment significantly increased activity of AP1 and decreased activity of factors including NFκB, SP1, E2F, and SMAD3. In an in vivo xenograft model of TNBC, individual and combination treatments resulted in a decrease in both tumor volume and mitotic indices. Taken together, these studies highlight the potential of this novel drug combination, CYC065 and eribulin, to suppress the growth of TNBC cells in vitro and in vivo, warranting further clinical investigation.

Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFß signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFß pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFß signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.

A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle.

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ-organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

Breast cancer onset and disease progression have been linked to members of the TGFß superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFß in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin.

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFß superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.

Superoxide generated at mitochondrial complex III triggers acute responses to hypoxia in the pulmonary circulation.

RATIONALE: The role of reactive oxygen species (ROS) signaling in the O(2) sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses. OBJECTIVES: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA). METHODS: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC). MEASUREMENTS AND MAIN RESULTS: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca(2+)](i). In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca(2+)](i) of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice. CONCLUSIONS: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca(2+)](i) that cause acute hypoxic pulmonary vasoconstriction.

Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels.

AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKß abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKß.

Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.

RATIONALE: Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation. OBJECTIVE: This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells. METHODS AND RESULTS: We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs. CONCLUSIONS: These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.

Hypoxia inducible factor-alpha activation in lymphoma and relationship to the thioredoxin family.

Hypoxia inducible factors (HIFs) activate oncogenic pathways, while thioredoxins (Trx), including Trx1 and Trx reductases-1 and -2 (TrxR1 and TrxR2), promote HIF-alpha stabilization. In immunoblotting studies in lymphoma cell lines we found that Raji and SUDHL4 cells exhibited normoxic HIF-2alpha protein stabilization. Five cell lines showed increased TrxR1 expression, while only Namalwa, HF1 and SUDHL4 had Trx1 and TrxR2 activation. Tissue microarrays in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) identified different HIF expression among histological subgroups (e.g. 44% DLBCL vs. 11% of FL cases with moderate-to-high expression of HIF-1alpha and HIF-2alpha, P = 0.0017). These data demonstrate that HIF and the thioredoxin family are abnormally activated in lymphoma.

Communication between integrin receptors facilitates epicardial cell adhesion and matrix organization.

Formation of the epicardium requires interactions between alpha(4)beta(1) integrin, and the extracellular matrix. We investigated the role of other integrins expressed by epicardial cells. We detected transcripts for alpha(5), alpha(8), alpha(v), beta(1), beta(3), and beta(5) integrins in the chick proepicardial organ (PE). We demonstrate that alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3) integrins are expressed by chick epicardial mesothelial cells (EMCs). Migration of EMCs in vitro was reduced by RGD-containing peptides. Using adenoviruses expressing an antisense to chick alpha(4) (AdGFPalpha4AS), full-length (Adhalpha4V5), and C-terminal deleted alpha(4) (Adhalpha4DeltaCV5), we found that EMCs were less able to adhere to vitronectin and fibronectin(120) indicating that alpha(4)beta(1) plays a role in regulating EMC adhesion to ligands of alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3). In Adhalpha4DeltaCV5-infected EMCs, alpha(5)beta(1) was diminished in fibrillar adhesions and new FN matrix assembly was abnormal. We propose that cooperation between alpha(4)beta(1) and RGD integrins is important for EMC adhesion and subepicardial matrix formation.

VCAM-1 inhibits TGFbeta stimulated epithelial-mesenchymal transformation by modulating Rho activity and stabilizing intercellular adhesion in epicardial mesothelial cells.

Regulation of epithelial-mesenchymal transformation (EMT) is of central importance both in normal development and in disease. During heart development, cells of the superficial epicardial mesothelium undergo EMT to give rise to precursor cells of the coronary vasculature and cardiac fibroblasts. Here we report that the alpha(4)beta(1) integrin ligand, VCAM-1, inhibits EMT of chick epicardial mesothelial cells stimulated by TGFbeta isoforms. We further investigated the molecular basis of this inhibition using cultured chick embryonic and rat adult epicardial mesothelial cells. We observed that VCAM-1 increased cortical actin filaments at intercellular junctions and reduced stress fibers across epicardial cells. VCAM-1 inhibited stress fiber formation by TGFbeta1, TGFbeta2, TGFbeta3 and lysophosphatidic acid and altered Rho activity stimulated by TGFbeta3. This was accompanied by an increase in tyrosine phosphorylation of p190RhoGAP. All three TGFbeta isoforms weakened intercellular adhesion, reduced membrane localization of beta-catenin and E-cadherin and stimulated epicardial EMT in chick hearts. Each of these effects was restricted by simultaneous VCAM-1 treatment. Our data support the hypothesis that VCAM-1 can alter epicardial EMT at two key points: it limits Rho-dependent events such as stress fiber formation and it maintains the association of beta-catenin and E-cadherin with the adherens junction.