Publications by authors named "Yumiko Sakurai"

38 Publications

Platelet heterogeneity enhances blood clot volumetric contraction: An example of asynchrono-mechanical amplification.

Biomaterials 2021 Jul 23;274:120828. Epub 2021 Apr 23.

George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 801 Ferst Drive, Atlanta, GA, 30332-0405, USA. Electronic address:

Physiological processes such as blood clotting and wound healing as well as pathologies such as fibroses and musculoskeletal contractures, all involve biological materials composed of a contracting cellular population within a fibrous matrix, yet how the microscale interactions among the cells and the matrix lead to the resultant emergent behavior at the macroscale tissue level remains poorly understood. Platelets, the anucleate cell fragments that do not divide nor synthesize extracellular matrix, represent an ideal model to study such systems. During blood clot contraction, microscopic platelets actively pull fibers to shrink the macroscale clot to less than 10% of its initial volume. We discovered that platelets utilize a new emergent behavior, asynchrono-mechanical amplification, to enhanced volumetric material contraction and to magnify contractile forces. This behavior is triggered by the heterogeneity in the timing of a population of actuators. This result indicates that cell heterogeneity, often attributed to stochastic cell-to-cell variability, can carry an essential biophysical function, thereby highlighting the importance of considering 4 dimensions (space + time) in cell-matrix biomaterials. This concept of amplification via heterogeneity can be harnessed to increase mechanical efficiency in diverse systems including implantable biomaterials, swarm robotics, and active polymer composites.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184644PMC
July 2021

A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy.

PLoS One 2020 9;15(11):e0241869. Epub 2020 Nov 9.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Chronic enteropathy associated with SLCO2A1 gene (CEAS) is caused by loss-of-function mutations in SLCO2A1, which encodes a prostaglandin (PG) transporter. In this study, we report a sibling case of CEAS with a novel pathogenic variant of the SLCO2A1 gene. Compound heterozygous variants in SLCO2A1 were identified in an 8-year-old boy and 12-year-old girl, and multiple chronic nonspecific ulcers were observed in the patients using capsule endoscopy. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported to be pathogenic, whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not yet been reported. The affected residue (p.Leu563Pro) is located in the 11th transmembrane domain (helix 11) of SLCO2A1. Because SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites were measured by liquid chromatography coupled to tandem mass spectrometry. The urinary tetranor-prostaglandin E metabolite levels in the patients were significantly higher than those in unaffected individuals. We established cell lines with doxycycline-inducible expression of wild type SLCO2A1 (WT-SLCO2A1) and the L563P mutant. Immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membranes of these cells. Cells expressing WT-SLCO2A1 exhibited time- and dose-dependent uptake of PGE2, while the mutant did not show any uptake activity. Residue L563 is very close to the putative substrate-binding site in SLCO2A1, R561 in helix 11. However, in a molecular model of SLCO2A1, the side chain of L563 projected outside of helix 11, indicating that L563 is likely not directly involved in substrate binding. Instead, the substitution of Pro may twist the helix and impair the transporter function. In summary, we identified a novel pathogenic variant of SLCO2A1 that caused loss-of-function and induced CEAS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241869PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652309PMC
January 2021

Getting a good view: imaging of platelets under flow.

Platelets 2020 Jul 28;31(5):570-579. Epub 2020 Feb 28.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University , Atlanta, GA, USA.

As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our imaging studies of platelets under flow.
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http://dx.doi.org/10.1080/09537104.2020.1732320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332395PMC
July 2020

Urinary angiotensinogen in pediatric urinary tract infection.

Pediatr Int 2019 Jul 23;61(7):712-714. Epub 2019 Jul 23.

Department of Pediatrics, Juntendo University, Tokyo, Japan.

Background: Urinary tract infection (UTI) is one of the most common diseases in children, and urinary angiotensinogen (U-AGT) is a new biomarker gathering attention in many renal diseases. U-AGT reflects intrarenal renin-angiotensin system (RAS) activity. We conducted a study to measure U-AGT in children <4 months old with UTI.

Methods: All children <4 months old who came to Toshima Hospital with fever between January 2015 and December 2015 were included. Patients were divided into a UTI group and a non-UTI group, and U-AGT was measured.

Results: Median U-AGT was higher in patients with UTI compared with patients without UTI: (0.56 ng/dL, range, 0.025-2.753 ng/dL vs 0.13 ng/dL, range, 0.008-1.697 ng/dL, respectively; P < 0.05).

Conclusions: U-AGT is elevated in UTI patients, and RAS activation may contribute to renal injury caused by UTI.
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http://dx.doi.org/10.1111/ped.13890DOI Listing
July 2019

The gene variants in Japanese children with idiopathic pancreatitis.

Hum Genome Var 2019 11;6:17. Epub 2019 Apr 11.

2Department of Genome Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535 Japan.

The cystic fibrosis transmembrane conductance regulator () gene has been reported as one of the pancreatitis susceptibility genes. Although many variants of have been reported in Caucasian patients, there are few data in Japanese patients. We aimed to survey variants in Japanese children with idiopathic pancreatitis. Twenty-eight Japanese paediatric patients with idiopathic pancreatitis were enroled, who were not previously diagnosed by genetic analysis of and . The entire gene was sequenced in the patients by combining LA-PCR and next-generation sequencing analysis. To determine a splice-affecting variant, expression was investigated in the nasal epithelial cells by RT-PCR. One (3.6%) and 15 (53.6%) of 28 patients had pathogenic and functionally affected variants in the gene, respectively. Two variants, p.Arg352Gln and p.Arg1453Trp, were found more frequently in the patients compared with one in Japanese healthy controls ( = 0.0078 and 0.044, respectively). We confirmed skipping of exon 10 in the nasal epithelial cells in one patient having a splice-affecting variant (c.1210-12 T(5)) in intron 9. Functionally affected variants of the gene are not so rare in Japanese paediatric patients with idiopathic pancreatitis. Surveying gene variants in a Japanese sample could help identify pancreatitis risk in these children.
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http://dx.doi.org/10.1038/s41439-019-0049-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459923PMC
April 2019

Microvasculature-on-a-chip for the long-term study of endothelial barrier dysfunction and microvascular obstruction in disease.

Nat Biomed Eng 2018 23;2:453-463. Epub 2018 Apr 23.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and hematologic disorders such as sickle cell disease (SCD) and malaria often lead to increased endothelial permeability, haemolysis, and microvascular obstruction. However, the associations among these pathological phenomena remain unknown. Here, we report a perfusable, endothelialized microvasculature-on-a-chip featuring an interpenetrating-polymer-network hydrogel that recapitulates the stiffness of blood-vessel intima, basement membrane self-deposition and self-healing endothelial barrier function for longer than 1 month. The microsystem enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found how extracellular heme, a hemolytic byproduct, induces delayed but reversible endothelial permeability in a dose-dependent manner, and demonstrate that endothelial interactions with SCD or malaria-infected erythrocytes cause reversible microchannel occlusion and increased in situ endothelial permeability. The microvasculature-on-a-chip enables mechanistic insight into the endothelial barrier dysfunction associated with SCD, malaria and other inflammatory and haematological diseases.
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http://dx.doi.org/10.1038/s41551-018-0224-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286070PMC
April 2018

Interdigitated microelectronic bandage augments hemostasis and clot formation at low applied voltage in vitro and in vivo.

Lab Chip 2018 09;18(19):2985-2993

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Hemorrhage or uncontrolled bleeding can arise either due to a medical condition or from a traumatic injury and are typically controlled with the application of a hemostatic agent. Hemostatic agents are currently derived from animal or human products, which carry risks of blood borne infections and immune dysregulation. Therefore, the need exists for novel biomedical therapies not derived from animal or human products to achieve hemostasis. Accordingly, we created an interdigitated microelectronic bandage that applies low voltage electrical stimulation to an injury site, resulting in faster clot formation without excessive heating, accelerated fibrin formation, and hemostasis overall. Our interdigitated microelectronic bandage found fibrin formed 1.5× faster in vitro. In vivo, total cessation of bleeding was 2.5× faster, resulting in 2× less blood loss. Electricity has been used in medical applications such as defibrillation, cauterization, and electrosurgery, but scant research has focused on hemostasis. Here we report a novel surface treatment using an interdigitated microelectronic device that creates rapid hemostasis in both in vitro and in vivo bleeding models with low applied voltages, representing a new and novel class of hemostatic agents that are electrically-based.
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http://dx.doi.org/10.1039/c8lc00573gDOI Listing
September 2018

A microengineered vascularized bleeding model that integrates the principal components of hemostasis.

Nat Commun 2018 02 6;9(1):509. Epub 2018 Feb 6.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 345 Ferst Drive, Atlanta, GA, 30332, USA.

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an "endothelialized" microfluidic system coupled with a microengineered pneumatic valve that induces a vascular "injury". With perfusion of whole blood, hemostatic plug formation is visualized and "in vitro bleeding time" is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.
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http://dx.doi.org/10.1038/s41467-018-02990-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802762PMC
February 2018

Extracellular fluid tonicity impacts sickle red blood cell deformability and adhesion.

Blood 2017 12 4;130(24):2654-2663. Epub 2017 Oct 4.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer & Blood Disorders Center, Emory University School of Medicine, Atlanta, GA.

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an "endothelialized" microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.
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http://dx.doi.org/10.1182/blood-2017-04-780635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731085PMC
December 2017

Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency.

Mol Ther 2017 10 8;25(10):2372-2382. Epub 2017 Jul 8.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA. Electronic address:

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1) and human (CD34) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628863PMC
October 2017

Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions.

Nat Commun 2017 06 8;8:15594. Epub 2017 Jun 8.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

The vascular endothelium presents a major transport barrier to drug delivery by only allowing selective extravasation of solutes and small molecules. Therefore, enhancing drug transport across the endothelial barrier has to rely on leaky vessels arising from disease states such as pathological angiogenesis and inflammatory response. Here we show that the permeability of vascular endothelium can be increased using an external magnetic field to temporarily disrupt endothelial adherens junctions through internalized iron oxide nanoparticles, activating the paracellular transport pathway and facilitating the local extravasation of circulating substances. This approach provides a physically controlled drug delivery method harnessing the biology of endothelial adherens junction and opens a new avenue for drug delivery in a broad range of biomedical research and therapeutic applications.
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http://dx.doi.org/10.1038/ncomms15594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472756PMC
June 2017

Platelet-Microcapsule Hybrids Leverage Contractile Force for Targeted Delivery of Hemostatic Agents.

ACS Nano 2017 06 5;11(6):5579-5589. Epub 2017 Jun 5.

Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta/Emory University School of Medicine , Atlanta, Georgia 30322, United States.

We report a cell-mediated, targeted drug delivery system utilizing polyelectrolyte multilayer capsules that hybridize with the patient's own platelets upon intravenous administration. The hybridized platelets function as the sensor and actuator for targeted drug delivery and controlled release in our system. These capsules are biochemically and mechanically tuned to enable platelet adhesion and capsule rupture upon platelet activation and contraction, enabling the targeted and controlled "burst" release of an encapsulated biotherapeutic. As platelets are the "first responders" in the blood clot formation process, this platelet-hybridized system is ideal for the targeted delivery of clot-augmenting biotherapeutics wherein immediate therapeutic efficacy is required. As proof-of-concept, we tailored this system to deliver the pro-clotting biotherapeutic factor VIII for hemophilia A patients that have developed inhibitory antifactor VIII antibodies. The polyelectrolyte multilayer capsules physically shield the encapsulated factor VIII from the patient's inhibitors during circulation, preserving its bioactivity until it is delivered at the target site via platelet contractile force. Using an in vitro microfluidic vascular injury model with factor VIII-inhibited blood, we demonstrate a 3.8× increase in induced fibrin formation using capsules loaded with factor VIII at a concentration an order of magnitude lower than that used in systemic delivery. We further demonstrate that clot formation occurs 18 min faster when factor VIII loaded capsules are used compared to systemic delivery at the same concentration. Because platelets are integral in the pathophysiology of thrombotic disorders, cancer, and innate immunity, this paradigm-shifting smart drug delivery system can be similarly applied to these diseases.
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http://dx.doi.org/10.1021/acsnano.7b00929DOI Listing
June 2017

Normal saline is associated with increased sickle red cell stiffness and prolonged transit times in a microfluidic model of the capillary system.

Microcirculation 2017 07;24(5)

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Center of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA.

Objective: Vaso-occlusive crisis (VOC) is a complex process that occurs in patients with sickle cell disease (SCD) and is often associated with pain and urgent hospitalization. A major instigator of VOC is microvascular obstruction by pathologically stiffened sickle red blood cells (RBCs), and thus, therapy relies heavily on optimizing intravenous fluid (IVF) hydration to increase RBC deformability. However, no evidence-based guidelines regarding the choice of IVF currently exist. We therefore analyzed alterations in biomechanical properties of sickle RBCs isolated from patients with homozygous SCD (hemoglobin SS) after exposure to different osmolarities of clinical IVF formulations.

Methods: Atomic force microscopy (AFM) was used to assess stiffness of RBCs after exposure to different IVFs. A microfluidic model of the human capillary system was used to assess transit time (TT) and propensity to occlusion after exposure to the different IVF formulations.

Results: Sickle RBCs exposed to normal saline (NS) had increased stiffness, TTs, and propensity to microchannel occlusion compared to other osmolarities.

Conclusion: NS, an IVF formulation often used to treat patients with SCD during VOC, may induce localized microvascular obstruction due to alterations of sickle RBC biomechanical properties.
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http://dx.doi.org/10.1111/micc.12353DOI Listing
July 2017

Single-platelet nanomechanics measured by high-throughput cytometry.

Nat Mater 2017 02 10;16(2):230-235. Epub 2016 Oct 10.

Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.
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http://dx.doi.org/10.1038/nmat4772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266633PMC
February 2017

Genetic Analysis of Japanese Children With Acute Recurrent and Chronic Pancreatitis.

J Pediatr Gastroenterol Nutr 2016 10;63(4):431-6

*Department of Pediatrics †Department of Gastroenterology, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Objectives: Causes of acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP) are sometimes difficult to determine in children. In such patients, genetic analysis may prove helpful. The present study analyzed mutations of cationic trypsinogen (PRSS1), serine protease inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC), and carboxypeptidase A1 (CPA1) and investigated the clinical features of children with these mutations.

Methods: Genetic analyses of mutations in these 4 genes were conducted in 128 patients with ARP or CP. Characteristics of the patients showing mutations were investigated using medical records.

Results: Fifty of the 128 (39.1%) subjects had at least 1 mutation (median age at onset, 7.6 years). Abdominal pain was the presenting symptom of pancreatitis in 48 of the 50 patients (96%). Fifteen of those 50 patients (30.0%) had a family history of pancreatitis. Gene mutations were present in PRSS1 in 26 patients, SPINK1 in 23, CTRC in 3, and CPA1 in 5. In the 31 patients with mutations in SPINK1, CTRC, or CPA1, 16 (51.6%) had homozygous or heterozygous mutations with other mutations. Three patients underwent surgery and another 4 patients underwent endoscopy to manage ARP or CP. Although 3 of the 7 patients complained of mild abdominal pain, none of those 7 patients experienced any obvious episode of ARP after treatment.

Conclusions: In pediatric patients with idiopathic ARP and CP, genetic analysis is useful for identifying the cause of pancreatitis. Early endoscopic or surgical treatment prevents ARP by extending the interval between episodes of pancreatitis in this population.
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http://dx.doi.org/10.1097/MPG.0000000000001320DOI Listing
October 2016

Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts.

Proc Natl Acad Sci U S A 2016 Feb 8;113(8):1987-92. Epub 2016 Feb 8.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA 30332; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322; Winship Cancer Institute, Emory University, Atlanta, GA 30322; Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332; Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332;

Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.
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http://dx.doi.org/10.1073/pnas.1508920113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776450PMC
February 2016

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition.

Blood 2015 Jul 11;126(4):531-8. Epub 2015 May 11.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Atlanta, GA; Department of Pediatrics, Emory University School of Medicine, Atlanta, GA; Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA; Winship Cancer Institute, Emory University, Atlanta, GA; Institute of Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA;

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.
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http://dx.doi.org/10.1182/blood-2014-11-607614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513253PMC
July 2015

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach.

Blood 2015 Aug 30;126(6):817-24. Epub 2015 Apr 30.

Wallace C Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA; Institute of Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA;

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an "endothelial-ized" microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model.
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http://dx.doi.org/10.1182/blood-2015-02-628594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528067PMC
August 2015

Platelet mechanosensing of collagen matrices.

PLoS One 2015 27;10(4):e0126624. Epub 2015 Apr 27.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia, United States of America; Winship Cancer Institute of Emory University, Atlanta, Georgia, United States of America.

During vascular injury, platelets adhere to exposed subendothelial proteins, such as collagen, on the blood vessel walls to trigger clot formation. Although the biochemical signalings of platelet-collagen interactions have been well characterized, little is known about the role microenvironmental biomechanical properties, such as vascular wall stiffness, may have on clot formation. To that end, we investigated how substrates of varying stiffness conjugated with the same concentration of Type I collagen affect platelet adhesion, spreading, and activation. Using collagen-conjugated polyacrylamide (PA) gels of different stiffnesses, we observed that platelets do in fact mechanotransduce the stiffness cues of collagen substrates, manifesting in increased platelet spreading on stiffer substrates. In addition, increasing substrate stiffness also increases phosphatidylserine exposure, a key aspect of platelet activation that initiates coagulation on the platelet surface. Mechanistically, these collagen substrate stiffness effects are mediated by extracellular calcium levels and actomyosin pathways driven by myosin light chain kinase but not Rho-associated protein kinase. Overall, our results improve our understanding of how the mechanics of different tissues and stroma affect clot formation, what role the increased vessel wall stiffness in atherosclerosis may directly have on thrombosis leading to heart attacks and strokes, and how age-related increased vessel wall stiffness affects hemostasis and thrombosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126624PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411076PMC
April 2016

Simplified prototyping of perfusable polystyrene microfluidics.

Biomicrofluidics 2014 Jul 30;8(4):046501. Epub 2014 Jul 30.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta , Georgia 30332, USA.

Cell culture in microfluidic systems has primarily been conducted in devices comprised of polydimethylsiloxane (PDMS) or other elastomers. As polystyrene (PS) is the most characterized and commonly used substrate material for cell culture, microfluidic cell culture would ideally be conducted in PS-based microsystems that also enable tight control of perfusion and hydrodynamic conditions, which are especially important for culture of vascular cell types. Here, we report a simple method to prototype perfusable PS microfluidics for endothelial cell culture under flow that can be fabricated using standard lithography and wet laboratory equipment to enable stable perfusion at shear stresses up to 300 dyn/cm(2) and pumping pressures up to 26 kPa for at least 100 h. This technique can also be extended to fabricate perfusable hybrid PS-PDMS microfluidics of which one application is for increased efficiency of viral transduction in non-adherent suspension cells by leveraging the high surface area to volume ratio of microfluidics and adhesion molecules that are optimized for PS substrates. These biologically compatible microfluidic devices can be made more accessible to biological-based laboratories through the outsourcing of lithography to various available microfluidic foundries.
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http://dx.doi.org/10.1063/1.4892035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189295PMC
July 2014

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation.

Proc Natl Acad Sci U S A 2014 Oct 22;111(40):14430-5. Epub 2014 Sep 22.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322; Winship Cancer Institute of Emory University, Atlanta, GA 30322; Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbβ3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.
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http://dx.doi.org/10.1073/pnas.1322917111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210024PMC
October 2014

Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo.

Stem Cell Reports 2014 Jul 6;3(1):60-72. Epub 2014 Jun 6.

Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA ; Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA.

Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic.
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http://dx.doi.org/10.1016/j.stemcr.2014.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110775PMC
July 2014

The platelet and the biophysical microenvironment: lessons from cellular mechanics.

Thromb Res 2014 Apr 4;133(4):532-7. Epub 2014 Jan 4.

Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA; Parker H. Petit Institute of Bioengineering & Bioscience, Georgia Institute of Technology, Atlanta, GA, USA; Winship Cancer Institute of Emory University, Atlanta, GA, USA. Electronic address:

While the role of platelets in hemostasis is well characterized from a biological perspective, the biophysical interactions between platelets and their mechanical microenvironment are relatively unstudied. The field of cellular mechanics has developed a number of approaches to study the effects of extracellular matrix (ECM)-derived mechanical forces on various cells, and has elucidated that integrin-cytoskeleton-mediated force transduction governs many cellular processes. As platelets adhere and spread via molecular machinery that is similar to that which enables other cells to mechanosense and mechanotransduce forces from their biophysical microenvironment, platelets too are likely governed by the same overarching mechanisms. Indeed, recent platelet mechanobiology studies have revealed that key aspects of platelet physiology and activation are regulated by the mechanical and spatial properties of the ECM microenvironment. At the same time, there are also key differences that make platelets unique in the world of cells-- their size, origin as megakaryocyte fragments, and unique αIIbβ3 integrin-- render their mechanosensing activities particularly interesting. The structurally "simple," anucleate nature of platelets coupled with their high actin concentration (20% of total protein) and integrin density [1] seem to make them ideal for mechanical force generation and transmission. Further studies will enhance our understanding of the role of platelet mechanobiology in hemostasis and thrombosis, potentially leading to new categories of diagnostics that investigate the mechanical properties of clots to determine bleeding risk, as well as therapies that target the mechanotransduction signaling pathway to alter the stability of clots.
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http://dx.doi.org/10.1016/j.thromres.2013.12.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959273PMC
April 2014

Biomechanics of haemostasis and thrombosis in health and disease: from the macro- to molecular scale.

J Cell Mol Med 2013 May 14;17(5):579-96. Epub 2013 Mar 14.

Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Although the processes of haemostasis and thrombosis have been studied extensively in the past several decades, much of the effort has been spent characterizing the biological and biochemical aspects of clotting. More recently, researchers have discovered that the function and physiology of blood cells and plasma proteins relevant in haematologic processes are mechanically, as well as biologically, regulated. This is not entirely surprising considering the extremely dynamic fluidic environment that these blood components exist in. Other cells in the body such as fibroblasts and endothelial cells have been found to biologically respond to their physical and mechanical environments, affecting aspects of cellular physiology as diverse as cytoskeletal architecture to gene expression to alterations of vital signalling pathways. In the circulation, blood cells and plasma proteins are constantly exposed to forces while they, in turn, also exert forces to regulate clot formation. These mechanical factors lead to biochemical and biomechanical changes on the macro- to molecular scale. Likewise, biochemical and biomechanical alterations in the microenvironment can ultimately impact the mechanical regulation of clot formation. The ways in which these factors all balance each other can be the difference between haemostasis and thrombosis. Here, we review how the biomechanics of blood cells intimately interact with the cellular and molecular biology to regulate haemostasis and thrombosis in the context of health and disease from the macro- to molecular scale. We will also show how these biomechanical forces in the context of haemostasis and thrombosis have been replicated or measured in vitro.
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http://dx.doi.org/10.1111/jcmm.12041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822810PMC
May 2013

Endothelialized microfluidics for studying microvascular interactions in hematologic diseases.

J Vis Exp 2012 Jun 22(64). Epub 2012 Jun 22.

Department of Pediatrics, Emory University School of Medicine, Georgia, USA. .

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales (1,2). In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions(3-6). As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions(6-13).However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature. Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.
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http://dx.doi.org/10.3791/3958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471282PMC
June 2012

Microenvironmental geometry guides platelet adhesion and spreading: a quantitative analysis at the single cell level.

PLoS One 2011 20;6(10):e26437. Epub 2011 Oct 20.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States of America.

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 µm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 µm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197646PMC
February 2012

Heat shock proteins and superantigenic properties of bacteria from the gastrointestinal tract of patients with Kawasaki disease.

Immunology 2009 Dec;128(4):511-20

Department of Pediatrics, Juntendo University School of Medicine, Tokyo, Japan.

We previously suggested that gut bacteria may be involved in the onset of Kawasaki disease (KD). In this study, we evaluated the production of heat shock proteins (hsps) and superantigens (sAgs) by microorganisms isolated from the jejunal mucosa of 19 children with KD in the acute phase and from 15 age-matched control children. We identified 13 strains of Gram-negative microbes from patients with KD; these microbes produced large amounts of hsp60 and induced pro-inflammatory cytokine production by peripheral blood mononuclear cells. The Gram-negative microbes also elicited endogenous hsp60 production, leading to the secretion of anti-inflammatory intereukin-10 (IL-10). We also identified 18 strains of Gram-positive cocci that had superantigenic properties and which induced the expansion of Vbeta2 T cells in vitro. All bacteria identified in this study were antibiotic resistant. These data suggest that sAg and hsps produced by gut bacteria might be involved in KD.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792135PMC
http://dx.doi.org/10.1111/j.1365-2567.2009.03135.xDOI Listing
December 2009

Modulating the functional contributions of c-Myc to the human endothelial cell cyclic strain response.

J Vasc Res 2010 3;47(1):80-90. Epub 2009 Sep 3.

Bioengineering Program, Georgia Institute of Technology, Atlanta, GA 30332-0535, USA.

This study addresses whether pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction in human umbilical vein endothelial cells (HUVEC) or human aortic endothelial cells (HAEC). mRNA and protein expression of c-Myc under physiological (6-10%) and pathological cyclic strain conditions (20%) were studied. Both c-Myc mRNA and protein expression increased 2-3-fold in HUVEC cyclically strained at 20%. c-Myc protein increased 4-fold in HAEC. In HUVEC, expression of mRNA peaked at 1.5-2 h. Subsequently, the effect of modulating c-Myc on potential downstream gene targets was determined. A small molecular weight compound that binds to and stabilizes the silencer element in the c-Myc promoter attenuates cyclic strain-induced c-Myc transcription by about 50%. This compound also modulates c-Myc downstream gene targets that may be instrumental in induction of vascular disease. Cyclic strain-induced gene expression of vascular endothelial growth factor, proliferating cell nuclear antigen and heat shock protein 60 are attenuated by this compound. These results offer a possible mechanism and promising clinical treatment for vascular diseases initiated by increased cyclic strain.
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http://dx.doi.org/10.1159/000235928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855285PMC
December 2009