Pathologic Stresses Research Articles

Clinical Outcomes of Total Knee Arthroplasty With Concomitant Total Ankle Arthroplasty Versus Ankle Arthrodesis

Prior studies have demonstrated a high incidence of ankle osteoarthritis (OA) in patients undergoing total knee arthroplasty (TKA) as well as inferior outcomes in the setting of ankle OA or hindfoot malalignment. Little is known about the effect of the 2 most common surgical treatments for ankle OA, ankle arthrodesis and total ankle arthroplasty (TAA) on TKA. This hypothesis is that the preservation of ankle motion afforded by total ankle arthroplasty may reduce pathologic stresses across the knee joint. This study compares outcomes of patients who underwent both TKA and TAA versus those that underwent TKA and ankle arthrodesis. We retrospectively reviewed a cohort of patients who had undergone TKA and either TAA or ankle arthrodesis at this institution, examining knee injury and OA outcome scores, foot and ankle ability measure scores, revision surgery, knee range of motion, and pain. There were 69 eligible subjects, 13 who had undergone total knee arthroplasty and total ankle arthroplasty and 56 who had undergone TKA and ankle arthrodesis. No significant differences were observed in KOOS Jr scores, FAAM scores, incidence of revision, knee range of motion, or pain at final follow up (p > .05). Mean follow-up time was 46 months after both surgeries were completed. Equivalent outcomes were observed between the 2 groups. The presence of a TKA should not alter the indications for treatment of ankle OA with TAA versus arthrodesis. Further studies are needed as these relatively rare concomitant procedures are likely to become more common in the future.

Neuregulins: protective and reparative growth factors in multiple forms of cardiovascular disease.

Neuregulins (NRGs) are protein ligands that act through ErbB receptor tyrosine kinases to regulate tissue morphogenesis, plasticity, and adaptive responses to physiologic needs in multiple tissues, including the heart and circulatory system. The role of NRG/ErbB signaling in cardiovascular biology, and how it responds to physiologic and pathologic stresses is a rapidly evolving field. While initial concepts focused on the role that NRG may play in regulating cardiac myocyte responses, including cell survival, growth, adaptation to stress, and proliferation, emerging data support a broader role for NRGs in the regulation of metabolism, inflammation, and fibrosis in response to injury. The constellation of effects modulated by NRGs may account for the findings that two distinct forms of recombinant NRG-1 have beneficial effects on cardiac function in humans with systolic heart failure. NRG-4 has recently emerged as an adipokine with similar potential to regulate cardiovascular responses to inflammation and injury. Beyond systolic heart failure, NRGs appear to have beneficial effects in diastolic heart failure, prevention of atherosclerosis, preventing adverse effects on diabetes on the heart and vasculature, including atherosclerosis, as well as the cardiac dysfunction associated with sepsis. Collectively, this literature supports the further examination of how this developmentally critical signaling system functions and how it might be leveraged to treat cardiovascular disease.

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.

ESCRT-III-driven piecemeal micro-ER-phagy remodels the ER during recovery from ER stress

The endoplasmic reticulum (ER) produces about 40% of the nucleated cell’s proteome. ER size and content in molecular chaperones increase upon physiologic and pathologic stresses on activation of unfolded protein responses (UPR). On stress resolution, the mammalian ER is remodeled to pre-stress, physiologic size and function on activation of the LC3-binding activity of the translocon component SEC62. This elicits recov-ER-phagy, i.e., the delivery of the excess ER generated during the phase of stress to endolysosomes (EL) for clearance. Here, ultrastructural and genetic analyses reveal that recov-ER-phagy entails the LC3 lipidation machinery and proceeds via piecemeal micro-ER-phagy, where RAB7/LAMP1-positive EL directly engulf excess ER in processes that rely on the Endosomal Sorting Complex Required for Transport (ESCRT)-III component CHMP4B and the accessory AAA+ ATPase VPS4A. Thus, ESCRT-III-driven micro-ER-phagy emerges as a key catabolic pathway activated to remodel the mammalian ER on recovery from ER stress.

Targeting the miR-665-3p-ATG4B-autophagy axis relieves inflammation and apoptosis in intestinal ischemia/reperfusion

Autophagy is an essential cytoprotective response against pathologic stresses that selectively degrades damaged cellular components. Impaired autophagy contributes to organ injury in multiple diseases, including ischemia/reperfusion (I/R), but the exact mechanism by which impaired autophagy is regulated remains unclear. Several researchers have demonstrated that microRNAs (miRNAs) negatively regulate autophagy by targeting autophagy-related genes (ATGs). Therefore, the effect of ATG-related miRNAs on I/R remains a promising research avenue. In our study, we found that autophagy flux is impaired during intestinal I/R. A miRNA microarray analysis showed that miR-665-3p was highly expressed in the I/R group, which was confirmed by qRT-PCR. Then, we predicted and proved that miR-665-3p negatively regulates ATG4B expression in Caco-2 and IEC-6 cells. In ileum biopsy samples from patients with intestinal infarction, there was an inverse correlation between miR-665-3p and ATG4B expression, which supports the in vitro findings. Moreover, based on miR-665-3p regulation of autophagy in response to hypoxia/reoxygenation in vitro, gain-of-function and loss-of-function approaches were used to investigate the therapeutic potential of miR-665-3p. Additionally, we provide evidence that ATG4B is indispensable for protection upon inhibition of miR-665-3p. Finally, we observed that locked nucleic acid-modified inhibition of miR-665-3p in vivo alleviates I/R-induced systemic inflammation and apoptosis via recovery of autophagic flux. Our study highlights miR-665-3p as a novel small molecule that regulates autophagy by targeting ATG4B, suggesting that miR-665-3p inhibition may be a potential therapeutic approach against inflammation and apoptosis for the clinical treatment of intestinal I/R.

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy

The mitochondrial Ca2+ uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca2+ influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca2+ uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to β-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca2+ uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of β-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with β-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy.

Abstract 431: Mitochondrial Fission in the Heart is an Adaptation to Increased Energetic Demands: The Role of Physiologic Fragmentation

Mitochondria play a dual role in the heart, responsible for meeting energetic demands and regulating cell death. Current paradigms hold that mitochondrial fission and fragmentation are the result of pathologic stresses such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in cardiac impairment, suggesting that fission is important for maintaining normal mitochondrial function. In this study, we identify a novel role for mitochondrial fragmentation as a normal physiological adaptation to increased energetic demand. Using two models of exercise, we demonstrate that “physiologic” mitochondrial fragmentation occurs, results in enhanced mitochondrial function, and is mediated through beta 1-adrenergic receptor signaling. Similar to pathologic fragmentation, physiologic fragmentation is induced by activation of Drp1; however, unlike pathologic fragmentation, membrane potential is maintained and regulators of mitophagy are downregulated. To confirm the role of fragmentation as a physiological adaptation to exercise, we inhibited the pro-fission mediator Drp1 in mice using the peptide inhibitor P110 and had mice undergo exercise. Mice treated with P110 had significantly decreased exercise capacity, decreased fragmentation and inactive Drp1 vs controls. To further confirm these findings, we generated cardiac-specific Drp1 KO mice and had them undergo exercise. Mice with cardiac specific Drp1 KO had significantly decreased exercise capacity and abnormally large mitochondria compared to controls. These findings indicate the requirement for physiological mitochondrial fragmentation to meet the energetic demands of exercise and support the still evolving conceptual framework, where fragmentation plays a role in the balance between mitochondrial maintenance of normal physiology and response to disease.

2015 AHA Late-Breaking Basic Science Abstracts.

### 23060 ### Validation Of Circulating MicroRnas As Biomarkers In Heart Failure In Two Large Independent Cohorts Antoni Bayes-Genis1, David Lanfear2, Maurice de Ronde3, Josep Lupon1, Joost Leenders4, Elisabet Zamora1, L. Keoki Williams2, De Antonio Marta5, Zhen Liu4, Koos Zwinderman3, Sara-Joan Pinto-Sietsma3, Yigal Pinto3; 1Universitat Autonoma de Barcelona, Barcelona, Spain; 2Henry Ford Hospital, Detroit, MI; 3Academic Med Cntr, Univ of Amsterdam, Amsterdam, Netherlands; 4Mirnext BV, Amsterdam, Netherlands; 5Germans Trias i Pujol Hosp, Badalona, Spain Background: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF. Methods: We measured 15 circmiRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification signals. Results: In general, between 20–40% of measurements were “invalid”, while miR-499a\_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a\_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II). …

Deficits in Hip-Ankle Coordination in Female Athletes who Suffer a Second Anterior Cruciate Ligament (ACL) Injury after ACL Reconstruction and Return to Sport

Objectives:Athletes who return to sport after ACL reconstruction (ACLR) are at increased risk of future ACL injury. Proprioceptive deficits at the knee, known to be present at thetime of ACL injury, may persist for up to 2 years after ACLR. Coordinated movements of the hip and ankle are critical in the absence of normal knee proprioception during dynamic athletic movements. Failure to optimally position the knee may make the passive structures susceptible to pathologic stresses that increase the risk of subsequent ligament or graft failure. The purpose of this work was to prospectively determine if altered lower extremity coordination patterns exist in female athletes, who go on to suffer a second ACL injury to either limb, after ACLR and return to sport (RTS). The study tested the hypothesis that female athletes who sustained a second ACL injury would demonstrate altered lower extremity coordination patterns indicative of persistent neuromuscular deficits at the time of return to sport compared to female athletes who would not subsequently sustain a second ACL injury.Methods:Sixty-one female athletes, who sustained an ACL injury, underwent ACLR, completed rehabilitation and were medically cleared for RTS were enrolled in this study. Hip-ankle coordination was assessed on all athletes prior to RTS as they performed a dynamic postural coordination task. The task required participants to stand on a single leg and track the anterior-posterior (AP) movement of an oscillating 3-D square target displayed on a computer monitor. Participants tracked the target with the head so as to maintain a constant perceived distance between their head and the target by matching the amplitude and frequency of the target oscillations. Fourteen patients sustained a second ACL injury within 12 months of RTS (ACLR2). Fourteen matched subjects after ACLR, who did not suffer a second ACL injury (ACLR1), were selected for comparative analysis. Cross-recurrence quantification analysis (Figure 1) was used to characterize hip-ankle coordination patterns through the variable cross-maxline (CML). A group (ACLR1 vs. ACLR2) X target speed (slow vs. fast) X leg (affected vs. unaffected) mixed-model analysis of variance was used to identify coordination differences.Results:A significant main effect of group was observed (p = 0.02), and indicated that the ACLR1 group exhibited more stable hip-ankle coordination overall (M = 166.2 ± 18.9) compared to the ACLR2 group (M = 108.4 ± 10.1), irrespective of target speed or tested leg. A leg x group interaction was also observed (p = .04). A Mann-Whitney test was employed due to unequal variances between groups, and indicated that the affected leg of the ACLR1 group exhibited more stable coordination (M = 187.1 ± 23.3)compared to the affected leg of the ACLR2 group (M = 110.13 ± 9.8), p = 0.03.Conclusion:Hip-ankle coordination was altered in female athletes who subsequently sustain a second ACL injury after initial ACLR and RTS. Failure to appropriately coordinate lower extremity movement between the adjoining proximal and distal hip and ankle in the absence of normal knee proprioception, may place the knee in a high-risk position and increase the likelihood of a second ACL injury in this population.

Regulation of Ischemic Neuronal Death by E2F4-p130 Protein Complexes

Inappropriate activation of cell cycle proteins, in particular cyclin D/Cdk4, is implicated in neuronal death induced by various pathologic stresses, including DNA damage and ischemia. Key targets of Cdk4 in proliferating cells include members of the E2F transcription factors, which mediate the expression of cell cycle proteins as well as death-inducing genes. However, the presence of multiple E2F family members complicates our understanding of their role in death. We focused on whether E2F4, an E2F member believed to exhibit crucial control over the maintenance of a differentiated state of neurons, may be critical in ischemic neuronal death. We observed that, in contrast to E2F1 and E2F3, which sensitize to death, E2F4 plays a crucial protective role in neuronal death evoked by DNA damage, hypoxia, and global ischemic insult both in vitro and in vivo. E2F4 occupies promoter regions of proapoptotic factors, such as B-Myb, under basal conditions. Following stress exposure, E2F4-p130 complexes are lost rapidly along with the presence of E2F4 at E2F-containing B-Myb promoter sites. In contrast, the presence of E2F1 at B-Myb sites increases with stress. Furthermore, B-Myb and C-Myb expression increases with ischemic insult. Taken together, we propose a model by which E2F4 plays a protective role in neurons from ischemic insult by forming repressive complexes that prevent prodeath factors such as Myb from being expressed.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

Adult Renal Mesenchymal Stem Cell–Like Cells Contribute to Juxtaglomerular Cell Recruitment

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44(+) mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44(+) MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.

Autophagy as a Stress-Response and Quality-Control Mechanism: Implications for Cell Injury and Human Disease

Autophagy, a vital catabolic process that degrades cytoplasmic components within the lysosome, is an essential cytoprotective response to pathologic stresses that occur during diseases such as cancer, ischemia, and infection. In addition to its role as a stress-response pathway, autophagy plays an essential quality-control function in the cell by promoting basal turnover of long-lived proteins and organelles, as well as by selectively degrading damaged cellular components. This homeostatic function protects against a wide variety of diseases, including neurodegeneration, myopathy, liver disease, and diabetes. This review discusses our current understanding of these two principal functions of autophagy and describes in detail how alterations in autophagy promote human disease.

Effects of low intensity ultrasound (LIUS) on the macular degeneration model in vitro

AbstractPurpose The retinal pigment epithelium (RPE) is compose of monolayer of tightly connected pigmented cells and supports the function of the normal vision function. Structural deformation and/or inflammation in RPE are commonly accompanied with and in many cases severely aggravate macular degeneration. We have reported previously low‐intensity ultrasound (LIUS) of less than 1 mW/cm^2 shows cyto‐protective and anti‐inflammatory effects on chondrocytes and cartilage tissue under pathologic environments. The aim of this study is to investigate the effects of LIUS on RPE cells in macular degeneration models in vitro.Methods ARPE‐19 cells were grown on a plastic dish or permeable transwell inserts and were treated with various damaging agents such as PlGF (an agonist of VEGF receptor), NaCN and H2O2. Then, cells were untreated or treated with varying intensities of LIUS with a maximum of 200 mW/cm^2 once for 20 min a day. Viability of cells was examined with Wst‐1 and TUNEL assays. Permeability of the cell monolayer on transwell membrane was tested by trans‐epithelial electrical resistance (TEER) system. JC‐1 staining was also performed to measure mitochondrial activity in cells.Results Cell damaging agents increased cell death and permeability of cell monolayer in a dose‐dependent dose manner. LIUS treatment significantly reduced both of the cell death and permeability. Changes in the mitochondrial membrane potential were also decreased by LIUS.Conclusion This study demonstrated that LIUS inhibits cell death of ARPE‐19 by various pathologic stresses, thereby might be a promising treatment for RPE under macular degeneration.

Catabolism of Branched-Chain Amino Acids in Heart Failure: Insights from Genetic Models

Genetic defects in amino acid metabolism are major causes of newborn diseases that often lead to abnormal development and function of the central nervous system. Their direct impact on cardiac development and function has rarely been investigated. Recently, the authors have established that a mitochondrial targeted 2C-type ser/thr protein phosphatase, PP2Cm, is the endogenous phosphatase of the branched-chain alpha keto acid-dehydrogenase complex (BCKD) and functions as a key regulator in branched-chain amino acid catabolism and homeostasis. Genetic inactivation of PP2Cm in mice leads to significant elevation in plasma concentrations of branched-chain amino acids and branched-chain keto acids at levels similar to those associated with intermediate mild forms of maple syrup urine disease. In addition to neuronal tissues, PP2Cm is highly expressed in cardiac muscle, and its expression is diminished in a heart under pathologic stresses. Whereas phenotypic features of heart failure are seen in PP2Cm-deficient zebra fish embryos, cardiac function in PP2Cm-null mice is compromised at a young age and deteriorates faster by mechanical overload. These observations suggest that the catabolism of branched-chain amino acids also has physiologic significance in maintaining normal cardiac function. Defects in PP2Cm-mediated catabolism of branched-chain amino acids can be a potential novel mechanism not only for maple syrup urine disease but also for congenital heart diseases and heart failure.

FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

The FOXO family of transcription factors elicits cell cycle arrest, apoptosis, and resistance to various physiologic and pathologic stresses relevant to sporadic cancer, such as DNA damage and oxidative stress. Although implicated as tumor suppressors, FOXO genetic inactivation has not been observed in human cancer. In an investigation of the two major types of non-small cell lung cancer, here, we identify the FOXO3 gene as a novel target of deletion in human lung adenocarcinoma (LAC). Biallelic or homozygous deletion (HD) of FOXO3 was detected in 8 of 33 (24.2%) mostly early-stage LAC of smokers. Another 60.6% of these tumors had losses of FOXO3 not reaching the level of HD (hereafter referred to as sub-HD). In contrast, no HD of FOXO3 was observed in 19 lung squamous cell carcinoma. Consistent with the deletion of FOXO3 were corresponding decreases in its mRNA and protein levels in LAC. The potential role of FOXO3 loss in LAC was also investigated. The carcinogen (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is strongly implicated as a cause of human lung cancer. Here, we show that FOXO3a is functionally activated and augments the level of caspase-dependent apoptosis in cells exposed to this DNA-damaging carcinogen. These results implicate FOXO3 as a suppressor of LAC carcinogenesis, a role frequently lost through gene deletion.

The Interaction of Von Willebrand Factor with GPIb-IX Initiates Platelet Apoptosis.

Abstract 3002Poster Board II-979 Background:The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates the adherence of platelets to sites of vascular injury, simultaneously triggers intracellular signaling events such as elevation of cytoplasmic calcium and activations of multiple protein kinase pathways which result in the activation of the ligand binding function of GPIIb/IIIa, leading to platelet activation and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the intracellular domain of GPIbalpha and play important roles in the regulation of platelet function. The signaling events elicited by GPIbalpha-VWF interaction, such as calcium mobilization and phosphatidylserine (PS) exposure are similar to those occurring during apoptosis. Particularly, the 14-3-3ζ binding domain of GPIbalpha has been reported to involve in the regulation of cell proliferations. However, it is still unclear whether the GPIbalpha-VWF interaction induces platelet apoptosis. Objectives:To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3ζ in apoptotic signaling. Methods:Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing GPIb-IX (1b9) interacted with VWF by flow cytometry or Western blotting. Results:Ristocetin induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF, however, the shear-induced apoptosis was eliminated by anti-GPIbalpha antibody AK-2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with truncation of the cytoplasmic domain of GPIbalpha or a serine-to-alanine mutation at 14-3-3ζ binding site in GPIbalpha. Conclusions:This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets and association of 14-3-3ζ with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. The finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases. The reagents that block 14-3-3ζ-GPIbalpha interaction might be potentially useful in platelet storage or anti-thrombocytopenia. Disclosures:No relevant conflicts of interest to declare.

The glycoprotein Ibalpha-von Willebrand factor interaction induces platelet apoptosis.

The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbalpha-VWF interaction. To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3zeta in apoptotic signaling. Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb-IX interacting with VWF by flow cytometry or western blotting. Ristocetin-induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIbalpha antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with GPIbalpha truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3zeta-binding site in GPIbalpha. This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3zeta with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.

Diminished GATA4 Protein Levels Contribute to Hyperglycemia-induced Cardiomyocyte Injury

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. The transcription factor GATA4 is essential for cardiac homeostasis, and its protein levels are dramatically reduced in the heart in response to diverse pathologic stresses. In this study, we investigated if hyperglycemia affects GATA4 expression in cardiomyocytes and if enhancing GATA4 signaling could attenuate hyperglycemia-induced cardiomyocyte injury. In cultured rat cardiomyocytes, high glucose (HG, 25 or 40 mm) markedly reduced GATA4 protein levels as compared with normal glucose (NG, 5.5 mm). Equal amount of mannitol did not affect GATA4 protein expression (NG, 100 +/- 12%; mannitol, 97 +/- 8%, versus HG, 43 +/- 16%, p < 0.05). The GATA4 mRNA content, either steady-state or polysome-associated, remained unchanged. HG-induced GATA4 reduction was reversed by MG262, a specific proteasome inhibitor. HG did not activate the ubiquitin proteasome system (UPS) in cardiomyocytes as indicated by a UPS reporter, nor did it increase the peptidase activities or protein expression of the proteasomal subunits. However, the mRNA levels of ubiquitin-protein isopeptide ligase (E3) carboxyl terminus of Hsp70-interacting protein (CHIP) were markedly increased in HG-treated cardiomyocytes. CHIP overexpression promoted GATA4 protein degradation, whereas small interfering RNA-mediated CHIP knockdown prevented HG-induced GATA4 depletion. Moreover, overexpression of GATA4 blocked HG-induced cardiomyocyte death. Also, GATA4 protein levels were diminished in the hearts of streptozotocin and db/db diabetic mice (44 +/- 7% and 67 +/- 13% of control, p < 0.05), which correlated with increased CHIP mRNA abundance. In summary, increased GATA4 protein degradation may be an important mechanism that contributes to hyperglycemic cardiotoxicity.

Derepression of pathological cardiac genes by members of the CaM kinase superfamily

In response to pathologic stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is characterized by myocyte hypertrophy, myocyte death and fibrosis, resulting in impaired cardiac function and heart failure. Cardiac remodeling is associated with derepression of genes that contribute to disease progression. This review focuses on evidence linking members of the Ca(2+)/calmodulin-dependent protein kinase (CaMK) superfamily, specifically CaMKII, protein kinase D (PKD) and microtubule associated kinase (MARK), to stress-induced derepression of pathological cardiac gene expression through their effects on class IIa histone deacetylases (HDACs).