Publications by authors named "Chieh-Yu Lin"

28 Publications

  • Page 1 of 1

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

bioRxiv 2020 Nov 5. Epub 2020 Nov 5.

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.
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http://dx.doi.org/10.1101/2020.11.04.364315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654892PMC
November 2020

Suppression of the Reactive Oxygen Response Alleviates Experimental Autoimmune Uveitis in Mice.

Int J Mol Sci 2020 May 5;21(9). Epub 2020 May 5.

Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.

Reactive oxygen species (ROS) are produced by host phagocytes and play an important role in antimicrobial actions against various pathogens. Autoimmune uveitis causes blindness and severe visual impairment in humans at all ages worldwide. However, the role of ROS in autoimmune uveitis remains unclear. We used ROS-deficient () mice to investigate the role of ROS in experimental autoimmune uveitis (EAU). Besides, we also used the antioxidant N-acetylcysteine (NAC) treatment to evaluate the effect of suppression of ROS on EAU in mice. The EAU disease scores of mice were significantly lower than those of wild-type mice. EAU induction increased the levels of cytokines (interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-12, IL-17, and tumor necrosis factor (TNF)-α) and chemokines (monocyte chemoattractant protein (MCP)-1) in the retinas of wild-type mice but not in those of mice. EAU induction enhanced the level of NF-κB activity in wild-type mice. However, the level of NF-κB activity in mice with EAU induction was low. Treatment with the antioxidant NAC also decreased the severity of EAU in mice with reduced levels of oxidative stress, inflammatory mediators, and NF-κB activation in the retina. We successfully revealed a novel role of ROS in the pathogenesis of EAU and suggest a potential antioxidant role for the treatment of autoimmune uveitis in the future.
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http://dx.doi.org/10.3390/ijms21093261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247341PMC
May 2020

CCR2 Positron Emission Tomography for the Assessment of Abdominal Aortic Aneurysm Inflammation and Rupture Prediction.

Circ Cardiovasc Imaging 2020 03 13;13(3):e009889. Epub 2020 Mar 13.

Department of Radiology (L.D., D.S., H.L., G.S.H., X.Z., R.L., J.Z., R.J.G., Y.L.), Washington University, St. Louis, MO.

Background: The monocyte chemoattractant protein-1/CCR2 (chemokine receptor 2) axis plays an important role in abdominal aortic aneurysm (AAA) pathogenesis, with effects on disease progression and anatomic stability. We assessed the expression of CCR2 in a rodent model and human tissues, using a targeted positron emission tomography radiotracer (Cu-DOTA-ECL1i).

Methods: AAAs were generated in Sprague-Dawley rats by exposing the infrarenal, intraluminal aorta to PPE (porcine pancreatic elastase) under pressure to induce aneurysmal degeneration. Heat-inactivated PPE was used to generate a sham operative control. Rat AAA rupture was stimulated by the administration of β-aminopropionitrile, a lysyl oxidase inhibitor. Biodistribution was performed in wild-type rats at 1 hour post tail vein injection of Cu-DOTA-ECL1i. Dynamic positron emission tomography/computed tomography imaging was performed in rats to determine the in vivo distribution of radiotracer.

Results: Biodistribution showed fast renal clearance. The localization of radiotracer uptake in AAA was verified with high-resolution computed tomography. At day 7 post-AAA induction, the radiotracer uptake (standardized uptake value [SUV]=0.91±0.25) was approximately twice that of sham-controls (SUV=0.47±0.10; <0.01). At 14 days post-AAA induction, radiotracer uptake by either group did not significantly change (AAA SUV=0.86±0.17 and sham-control SUV=0.46±0.10), independent of variations in aortic diameter. Competitive CCR2 receptor blocking significantly decreased AAA uptake (SUV=0.42±0.09). Tracer uptake in AAAs that subsequently ruptured (SUV=1.31±0.14; <0.005) demonstrated uptake nearly twice that of nonruptured AAAs (SUV=0.73±0.11). Histopathologic characterization of rat and human AAA tissues obtained from surgery revealed increased expression of CCR2 that was co-localized with CD68 macrophages. Ex vivo autoradiography demonstrated specific binding of Cu-DOTA-ECL1i to CCR2 in both rat and human aortic tissues.

Conclusions: CCR2 positron emission tomography is a promising new biomarker for the noninvasive assessment of AAA inflammation that may aid in associated rupture prediction.
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http://dx.doi.org/10.1161/CIRCIMAGING.119.009889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101060PMC
March 2020

Myocardial B cells are a subset of circulating lymphocytes with delayed transit through the heart.

JCI Insight 2020 02 13;5(3). Epub 2020 Feb 13.

Cardiovascular Division, Department of Medicine, and.

Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs, without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (>95%) of myocardial B cells remain intravascular, whereas few (<5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell-deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.
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http://dx.doi.org/10.1172/jci.insight.134700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098796PMC
February 2020

Heterogeneous Cellular Contributions to Elastic Laminae Formation in Arterial Wall Development.

Circ Res 2019 11 8;125(11):1006-1018. Epub 2019 Oct 8.

Department of Mechanical Engineering and Materials Science (M.C.S., J.Z.H., J.E.W.).

Rationale: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown.

Objective: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries.

Methods And Results: We used SMC- and EC-specific recombinase transgenes with a novel floxed allele to focus gene inactivation in mice. Inactivation of in SMCs using resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using or resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries.

Conclusions: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017653PMC
November 2019

Rosai-Dorfman Disease of the Breast With Variable IgG4+ Plasma Cells: A Diagnostic Mimicker of Other Malignant and Reactive Entities.

Am J Surg Pathol 2019 12;43(12):1653-1660

Department of Pathology, Stanford University, Stanford.

Rosai-Dorfman disease (RDD) is an uncommon disorder, characterized by an atypical expansion of histiocytes which classically shows emperipolesis and immunoreactivity with S-100 protein. RDD affects the lymph nodes as well as extranodal sites; however, RDD of the breast is exceptionally rare. Herein, we describe the histopathologic features of 22 cases of RDD occurring in the breast, with an emphasis on the differential diagnosis. All cases were notable for an exuberant lymphocytic infiltrate with and without germinal center formation, and the majority (19/22) showed numerous plasma cells: 5 to 132/high-power field (HPF). IgG and IgG4 immunohistochemical stains were available for 13 cases; in no instance were criteria for IgG4-related sclerosing disease met, though in a single case the IgG4/IgG ratio was increased to 25%. Sclerosis was present in the majority of cases (18/22), and was frequently prominent. RDD cells showing emperipolesis were present in all cases (22/22), and ranged from rare (<1/50 HPF) to numerous (>50/50 HPF). Two of the cases in our series were initially misdiagnosed as inflammatory myofibroblastic tumor and plasma cell mastitis with granulomatous inflammation. As emperipolesis can be indistinct, the presence of stromal fibrosis and a prominent lymphoplasmacytic inflammatory infiltrate should prompt a careful search for the characteristic histiocytes, which can be aided by the use of S-100 immunohistochemistry.
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http://dx.doi.org/10.1097/PAS.0000000000001347DOI Listing
December 2019

Genomic landscape of ductal carcinoma in situ and association with progression.

Breast Cancer Res Treat 2019 Nov 17;178(2):307-316. Epub 2019 Aug 17.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Purpose: The detection rate of breast ductal carcinoma in situ (DCIS) has increased significantly, raising the concern that DCIS is overdiagnosed and overtreated. Therefore, there is an unmet clinical need to better predict the risk of progression among DCIS patients. Our hypothesis is that by combining molecular signatures with clinicopathologic features, we can elucidate the biology of breast cancer progression, and risk-stratify patients with DCIS.

Methods: Targeted exon sequencing with a custom panel of 223 genes/regions was performed for 125 DCIS cases. Among them, 60 were from cases having concurrent or subsequent invasive breast cancer (IBC) (DCIS + IBC group), and 65 from cases with no IBC development over a median follow-up of 13 years (DCIS-only group). Copy number alterations in chromosome 1q32, 8q24, and 11q13 were analyzed using fluorescence in situ hybridization (FISH). Multivariable logistic regression models were fit to the outcome of DCIS progression to IBC as functions of demographic and clinical features.

Results: We observed recurrent variants of known IBC-related mutations, and the most commonly mutated genes in DCIS were PIK3CA (34.4%) and TP53 (18.4%). There was an inverse association between PIK3CA kinase domain mutations and progression (Odds Ratio [OR] 10.2, p < 0.05). Copy number variations in 1q32 and 8q24 were associated with progression (OR 9.3 and 46, respectively; both p < 0.05).

Conclusions: PIK3CA kinase domain mutations and the absence of copy number gains in DCIS are protective against progression to IBC. These results may guide efforts to distinguish low-risk from high-risk DCIS.
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http://dx.doi.org/10.1007/s10549-019-05401-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800639PMC
November 2019

Molecular profiling of clear cell adenocarcinoma of the urinary tract.

Virchows Arch 2019 Dec 2;475(6):727-734. Epub 2019 Aug 2.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Clear cell adenocarcinoma (CCA) of the urinary tract is a rare type of malignancy whose molecular profiles remain undefined. Here we reported an integrated clinicopathologic and molecular profiling analysis of four cases of clear cell adenocarcinoma arising in the urethra or the bladder. Utilizing a clinically validated 130-gene exon-sequencing assay, we identified recurrent pathogenic PIK3CA (p. E545K) and KRAS (p.G12D) variants in three of four (75%) of the cases. In addition, an APC variant (P.S2310X), a TP53 variant (p.R273C), and a MYC amplification event were identified. The only CCA case without either PIK3CA or KRAS variants has a distinct pathogenesis through BK virus, demonstrated by positive BK virus PCR and SV40 immunohistochemistry. The novel finding of recurrent variants in the PI3K/AKT/mTOR pathway provides not only insights into oncogenesis but also potential clinical therapeutic targets for patients with clear cell adenocarcinoma of the urinary tract.
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http://dx.doi.org/10.1007/s00428-019-02634-5DOI Listing
December 2019

Granular Cell Pituitary Tumor in a Patient with Multiple Endocrine Neoplasia-1.

Cureus 2019 Apr 25;11(4):e4541. Epub 2019 Apr 25.

Neurosurgery, Stanford University School of Medicine, Stanford, USA.

Multiple endocrine neoplasia type 1 (MEN-1) is an autosomal dominant disorder characterized by parathyroid, pancreatic islet, and pituitary tumors. Approximately 40% of MEN-1 patients harbor a pituitary adenoma. Separately, granular cell tumors (GCTs) of the sellar/parasellar region are an exceedingly rare clinical entity with less than 100 reported cases in the literature. These slow-growing, often asymptomatic lesions are difficult to diagnose and may mimic pituitary adenoma, Rathke cleft cyst, or other sellar/supra-sellar pathology. There is no known association with MEN-1 or any other familial syndrome. A 36-year-old neurologically normal woman with known MEN-1 underwent a screening magnetic resonance imaging (MRI) scan which revealed a 10 mm x 6 mm x 7 mm sellar/suprasellar lesion. She underwent endoscopic endonasal transsphenoidal resection. Subsequent neuropathological analysis was consistent with GCT of the pituitary gland. Here we describe the first report to our knowledge of a GCT of the pituitary gland occurring in a patient with MEN-1.
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http://dx.doi.org/10.7759/cureus.4541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592835PMC
April 2019

Adrenal Myelolipomas Involved by Plasma Cell Myeloma.

Am J Clin Pathol 2018 Oct;150(5):406-414

Department of Pathology, Stanford University School of Medicine, Stanford, CA.

Objectives: To report the presence and evaluate the frequency of plasma cell neoplasms within adrenal myelolipomas.

Methods: Adrenal myelolipomas within our institution were reviewed for the presence of hematologic neoplasia, and a review of the literature was performed.

Results: Two (9%) of 23 adrenal myelolipomas were involved by plasma cell myeloma. The patients were 71 and 81 years old, one woman and one man, with tumors measuring 7 cm and 8.5 cm, respectively. Both tumors contained large aggregates of dysplastic plasma cells occupying at least one ×10 field and demonstrated light chain restriction. Neither had an established diagnosis of plasma cell neoplasm previously. After receiving therapy, one patient exhibited a stable clinical course 1 year after diagnosis while the other died of disease 3 years later.

Conclusions: We report the first two cases of adrenal myelolipoma involved by plasma cell myeloma, a rare and subtle finding that has significant clinical implications.
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http://dx.doi.org/10.1093/ajcp/aqy068DOI Listing
October 2018

Genetics of the extracellular matrix in aortic aneurysmal diseases.

Matrix Biol 2018 10 12;71-72:128-143. Epub 2018 Apr 12.

Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA; McDonell Genome Institute, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

Aortic aneurysms are morbid conditions that can lead to rupture or dissection and are categorized as thoracic (TAA) or abdominal aortic aneurysms (AAA) depending on their location. While AAA shares overlapping risk factors with atherosclerotic cardiovascular disease, TAA exhibits strong heritability. Human genetic studies in the past two decades have successfully identified numerous genes involved in both familial and sporadic forms of aortic aneurysm. In this review we will discuss the genetic basis of aortic aneurysm, focusing on the extracellular matrix and how insights from these studies have informed our understanding of human biology and disease pathogenesis.
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http://dx.doi.org/10.1016/j.matbio.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146054PMC
October 2018

Comprehensive Genomic Profiling of Malignant Effusions in Patients with Metastatic Lung Adenocarcinoma.

J Mol Diagn 2018 03 19;20(2):184-194. Epub 2017 Dec 19.

Department of Pathology, Stanford University School of Medicine, Stanford, California. Electronic address:

Cytology samples are increasingly used for comprehensive molecular testing. Although fine-needle aspirates are adequate substrates for high-throughput sequencing, the suitability of malignant body fluids remains largely unexplored. We investigated the adequacy and utility of performing targeted next-generation sequencing (NGS) on malignant effusions from patients with metastatic lung adenocarcinoma. Thirty-two effusion samples submitted for hybrid capture-based NGS using a clinically validated solid tumor genotyping panel were examined. All cases showed ≥5% tumor cellularity; however, 28 (88%) provided sufficient DNA for NGS (≥1 ng/μL). The sequencing reads showed satisfactory quality control statistics, and the variant allele frequencies were correlated with tumor cellularity. Furthermore, pathogenic or likely pathogenic genomic alterations were identified in 26 of 28 samples (93%), whereas clinically actionable alterations were present in 18 (64%). Notably, nine patients had additional molecular testing performed on preceding or subsequent biopsy specimens, and the results across multiple samples were compared. In two patients, the NGS-based fluid analysis identified clinically actionable alterations that were not detected by other hotspot testing. In four patients treated with tyrosine kinase inhibitors, malignant fluid sequencing confirmed driver alterations from prior testing and revealed new resistance mechanisms. Hence, given adequate DNA input and tumor cellularity, comprehensive genomic profiling of malignant effusions may be used to establish mutational status at diagnosis and inform treatment resistance during targeted therapy.
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http://dx.doi.org/10.1016/j.jmoldx.2017.10.007DOI Listing
March 2018

Will oncotype DX DCIS testing guide therapy? A single-institution correlation of oncotype DX DCIS results with histopathologic findings and clinical management decisions.

Mod Pathol 2018 04 15;31(4):562-568. Epub 2017 Dec 15.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Given the increased detection rates of ductal carcinoma in situ (DCIS) and the limited overall survival benefit from adjuvant breast irradiation after breast-conserving surgery, there is interest in identifying subsets of patients who have low rates of ipsilateral breast tumor recurrence such that they might safely forgo radiation. The Oncotype DCIS score is a reverse transcription-PCR (RT-PCR)-based assay that was validated to predict which DCIS cases are most likely to recur. Clinically, these results may be used to assist in selecting which patients with DCIS might safely forgo radiation therapy after breast-conserving surgery; however, little is currently published on how this test is being used in practice. Our study examines traditional histopathologic features used in predicting DCIS risk with Oncotype DCIS results and how these results affect clinical decision-making at our academic institution. Histopathologic features and management decisions for 37 cases with Oncotype DCIS results over the past 4 years were collected. Necrosis, high nuclear grade, biopsy site change, estrogen receptor and progesterone receptor positivity <90% on immunohistochemistry, and Van Nuys Prognostic Index score of 8 or greater were significant predictors of an intermediate-high recurrence score on multivariate regression analysis (P<0.02). Low Oncotype DCIS scores and low nuclear grade were associated with lower rate of radiation therapy (P<0.008). There were seven cases (19%) with Oncotype DCIS results that we considered unexpected in relation to the histopathologic findings (ie, high nuclear grade with comedonecrosis and a low Oncotype score, or hormone receptor discrepancies). Overall, pathologic features correlate with Oncotype DCIS scores but unexpected results do occur, making individual recommendations sometimes challenging.
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http://dx.doi.org/10.1038/modpathol.2017.172DOI Listing
April 2018

Regional Variability in Percentage of Breast Cancers Reported as Positive for HER2 in California: Implications of Patient Demographics on Laboratory Benchmarks.

Am J Clin Pathol 2017 Sep;148(3):199-207

Department of Pathology.

Objectives: The expected regional variability in percent human epidermal growth factor receptor 2 (HER2)-positive breast cancers is not currently clear.

Methods: Data from the 2006 to 2011 California Cancer Registry were examined by county and health service area. The influence of demographic and pathologic features was used in a multivariable logistic regression model to compare expected with observed HER2-positive percentages by region.

Results: There was significant geographic variation by California counties (11.6%-26%). The reported HER2-positive percentage was higher when the population had higher stage, tumor size, grade, percent estrogen receptor negative, younger age, or lower socioeconomic status. Ethnic distribution of the population also influenced HER2-positive percentages. Using a multivariable logistic regression model, most regions had expected values based on their population characteristics; however, "outlier" regions were identified.

Conclusions: These results deepen our understanding of population characteristics' influence on the distribution of HER2-positive breast cancers. Taking these factors into account can be useful when setting laboratory benchmarks and assessing test quality.
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http://dx.doi.org/10.1093/ajcp/aqx063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848426PMC
September 2017

Breast cancer stem cells: are we ready to go from bench to bedside?

Histopathology 2016 Jan;68(1):119-37

Department of Pathology, Stanford University, Stanford, CA, USA.

Since the discovery of breast cancer stem cells (BCSCs) more than 10 years ago, a body of exciting research has developed. The intrinsic properties of BCSCs, including self-renewal and the ability to give rise to heterogeneous progeny, make BCSCs a likely source of tumour initiation, heterogeneity, progression and metastasis. BCSCs are also inherently resistant to conventional therapies and are therefore thought to contribute to disease recurrence. In this review, we will focus on both the challenges and recent advances in the characterization of BCSCs with respect to phenotype, molecular signature and their role in the behaviour of the different molecular subtypes of breast cancer. Of most importance is our ability to translate our growing knowledge base into the development of targeted therapies with the goal of reducing adverse outcomes in breast cancer patients.
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http://dx.doi.org/10.1111/his.12868DOI Listing
January 2016

A long noncoding RNA protects the heart from pathological hypertrophy.

Nature 2014 Oct 10;514(7520):102-106. Epub 2014 Aug 10.

Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine.

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA-chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized--but not naked--DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA-chromatin interaction.
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http://dx.doi.org/10.1038/nature13596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184960PMC
October 2014

Inappropriate p53 activation during development induces features of CHARGE syndrome.

Nature 2014 Oct 3;514(7521):228-32. Epub 2014 Aug 3.

1] Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, Stanford, California 94305, USA [2] Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA.

CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.
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http://dx.doi.org/10.1038/nature13585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192026PMC
October 2014

Preimplantation genetic diagnosis by fluorescence in situ hybridization of reciprocal and Robertsonian translocations.

Taiwan J Obstet Gynecol 2014 Mar;53(1):48-52

Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital and Medical College, TaoYuan, Taiwan. Electronic address:

Objective: The presence of reciprocal and Robertsonian chromosomal rearrangement is often related to recurrent miscarriage. Using preimplantation genetic diagnosis, the abortion rate can be decreased. Cases treated at our center were reviewed.

Materials And Methods: A retrospective analysis for either Robertsonian or reciprocal translocations was performed on all completed cycles of preimplantation genetic diagnosis at our center since the first reported case in 2004 until the end of 2010. Day 3 embryo biopsies were carried out, and the biopsied cell was checked by fluorescent in situ hybridization using relevant informative probes. Embryos with a normal or balanced translocation karyotype were transferred on Day 4.

Results: Thirty-eight preimplantation genetic diagnosis cycles involving 17 couples were completed. A total of 450 (82.6%) of the total oocytes were MII oocytes, and 158 (60.0%) of the two-pronuclei embryos were biopsied. In 41.4% of the fluorescent in situ hybridization analyses, the results were either normal or balanced. Embryos were transferred back after 21 cycles. Three babies were born from Robertsonian translocation carriers and another two from reciprocal translocation carriers. The miscarriage rate was 0%. Among the reciprocal translocation group, the live delivery rate was 8.3% per ovum pick-up cycle and 18.2% per embryo transfer cycle. Among the Robertsonian translocation group, the live delivery rate was 14.3% per ovum pick-up cycle and 20.0% per embryo transfer cycle.

Conclusion: There is a trend whereby the outcome for Robertsonian translocation group carriers is better than that for reciprocal translocation group carriers. Aneuploidy screening may possibly be added in order to improve the outcome, especially for individuals with an advanced maternal age. The emergence of an array-based technology should help improve this type of analysis.
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http://dx.doi.org/10.1016/j.tjog.2012.04.043DOI Listing
March 2014

Pbx1 activates Fgf10 in the mesenchyme of developing lungs.

Genesis 2014 May 14;52(5):399-407. Epub 2014 Mar 14.

Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California.

Insufficiency of surfactants is a core factor in respiratory distress syndrome, which causes apnea and neonatal death, particularly in preterm infants. Surfactant proteins are secreted by alveolar type II cells in the lung epithelium, the differentiation of which is regulated by Fgf10 elaborated by the adjacent mesenchyme. However, the molecular regulation of mesenchymal Fgf10 during lung development has not been fully understood. Here, we show that Pbx1, a homeodomain transcription factor, is required in the lung mesenchyme for the expression of Fgf10. Mouse embryos lacking Pbx1 in the lung mesenchyme show compact terminal saccules and perinatal lethality with failure of postnatal alveolar expansion. Mutant embryos had severely reduced expression of Fgf10 and surfactant genes (Spa, Spb, Spc, and Spd) that are essential for alveolar expansion for gas exchange at birth. Molecularly, Pbx1 directly binds to the Fgf10 promoter and cooperates with Meis and Hox proteins to transcriptionally activate Fgf10. Our results thus show how Pbx1 controls Fgf10 in the developing lung.
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http://dx.doi.org/10.1002/dvg.22764DOI Listing
May 2014

Epicardial calcineurin-NFAT signals through Smad2 to direct coronary smooth muscle cell and arterial wall development.

Cardiovasc Res 2014 Jan 14;101(1):120-9. Epub 2013 Aug 14.

Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

Aims: Congenital coronary artery anomalies produce serious events that include syncope, arrhythmias, myocardial infarction, or sudden death. Studying the mechanism of coronary development will contribute to the understanding of the disease and help design new diagnostic or therapeutic strategies. Here, we characterized a new calcineurin-NFAT signalling which specifically functions in the epicardium to regulate the development of smooth muscle wall of the coronary arteries.

Methods And Results: Using tissue-specific gene deletion, we found that calcineurin-NFAT signals in the embryonic epicardium to direct coronary smooth muscle cell development. The smooth muscle wall of coronary arteries fails to mature in mice with epicardial deletion of calcineurin B1 (Cnb1), and accordingly these mutant mice develop cardiac dysfunction with reduced exercise capacity. Inhibition of calcineurin at various developmental windows shows that calcineurin-NFAT signals within a narrow time window at embryonic Day 12.5-13.5 to regulate coronary smooth muscle cell development. Within the epicardium, NFAT transcriptionally activates the expression of Smad2, whose gene product is critical for transducing transforming growth factor β (TGFβ)-Alk5 signalling to control coronary development.

Conclusion: Our findings demonstrate new spatiotemporal and molecular actions of calcineurin-NFAT that dictate coronary arterial wall development and a new mechanism by which calcineurin-NFAT integrates with TGFβ signalling during embryonic development.
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http://dx.doi.org/10.1093/cvr/cvt197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868347PMC
January 2014

Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development.

Proc Natl Acad Sci U S A 2013 Jan 14;110(5):1738-43. Epub 2013 Jan 14.

Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21(cip1), to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.
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http://dx.doi.org/10.1073/pnas.1218072110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562770PMC
January 2013

Partitioning the heart: mechanisms of cardiac septation and valve development.

Development 2012 Sep;139(18):3277-99

Division of Cardiovascular Medicine, Department of Medicine, Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA.

Heart malformations are common congenital defects in humans. Many congenital heart defects involve anomalies in cardiac septation or valve development, and understanding the developmental mechanisms that underlie the formation of cardiac septal and valvular tissues thus has important implications for the diagnosis, prevention and treatment of congenital heart disease. The development of heart septa and valves involves multiple types of progenitor cells that arise either within or outside the heart. Here, we review the morphogenetic events and genetic networks that regulate spatiotemporal interactions between the cells that give rise to septal and valvular tissues and hence partition the heart.
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http://dx.doi.org/10.1242/dev.063495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424040PMC
September 2012

The secondary heart field is a new site of calcineurin/Nfatc1 signaling for semilunar valve development.

J Mol Cell Cardiol 2012 May 26;52(5):1096-102. Epub 2012 Jan 26.

Division of Cardiovascular Medicine, Department of Medicine, Stanford Cardiovascular Institute, Stanford University, Stanford, California 94305, USA.

Semilunar valve malformations are common human congenital heart defects. Bicuspid aortic valves occur in 2-3% of the population, and pulmonic valve stenosis constitutes 10% of all congenital heart disease in adults (Brickner et al., 2000) [1]. Semilunar valve defects cause valve regurgitation, stenosis, or calcification, leading to endocarditis or congestive heart failure. These complications often require prolonged medical treatment or surgical intervention. Despite the medical importance of valve disease, the regulatory pathways governing semilunar valve development are not entirely clear. In this report we investigated the spatiotemporal role of calcineurin/Nfatc1 signaling in semilunar valve development. We generated conditional knockout mice with calcineurin gene disrupted in various tissues during semilunar valve development. Our studies showed that calcineurin/Nfatc1 pathway signals in the secondary heart field (SHF) but not in the outflow tract myocardium or neural crest cells to regulate semilunar valve morphogenesis. Without SHF calcineurin/Nfatc1 signaling, the conal endocardial cushions-the site of prospective semilunar valve formation--first develop and then regress due to apoptosis, resulting in a striking phenotype with complete absence of the aortic and pulmonic valves, severe valve regurgitation, and perinatal lethality. This role of calcineurin/Nfatc1 signaling in the SHF is different from the requirement of calcineurin/Nfatc1 in the endocardium for semilunar valve formation (Chang et al., 2004) [2], indicating that calcineurin/Nfatc1 signals in multiple tissues to organize semilunar valve development. Also, our studies suggest distinct mechanisms of calcineurin/Nfat signaling for semilunar and atrioventricular valve morphogenesis. Therefore, we demonstrate a novel developmental mechanism in which calcineurin signals through Nfatc1 in the secondary heart field to promote semilunar valve morphogenesis, revealing a new supportive role of the secondary heart field for semilunar valve formation.
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http://dx.doi.org/10.1016/j.yjmcc.2012.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327781PMC
May 2012

Site-specific endometrial injury improves implantation and pregnancy in patients with repeated implantation failures.

Reprod Biol Endocrinol 2011 Oct 21;9:140. Epub 2011 Oct 21.

Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Medical Center, Chang Gung University, 5 Fu-Shin Street, Kweishan, Taoyuan, Taiwan.

Background: To test whether a site-specific hysteroscopic biopsy-induced injury in the endometrium during the controlled ovarian hyperstimulation cycle improves subsequent embryo implantation in patients with repeated implantation failure, a total of 30 patients who have had good responses to controlled ovulation stimulation but have failed to achieve pregnancy after two or more transfers of good-quality embryos were recruited in this prospective study.

Methods: A single, site-specific hysteroscopic biopsy-induced injury was generated on the posterior endometrium at midline 10-15 mm from the fundus during the D4-D7 period of the ongoing controlled ovarian hyperstimulation cycle in six patients.

Results: Patients received endometrial biopsy protocol achieved a pregnancy rate of 100%. By contrast, only 46% of patients with similar clinical characteristics (N = 24) achieved pregnancy without the hysteroscopic biopsy-induced endometrium injury (p < 0.05).

Conclusions: Our proof-of-concept study demonstrates that a site-specific hysteroscopic endometrium injury performed during the ongoing in vitro fertilization (IVF) cycle, instead of injuries received during prior cycles, significantly improves clinical outcomes in patients with repeated implantation failure.
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http://dx.doi.org/10.1186/1477-7827-9-140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210086PMC
October 2011

Rottlerin inhibits migration of follicular thyroid carcinoma cells by PKCdelta-independent destabilization of the focal adhesion complex.

J Cell Biochem 2010 May;110(2):428-37

Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.

This study examined the effect of rottlerin on the focal adhesion-mediated cell migration of CGTH W-2 human follicular thyroid carcinoma cells. Rottlerin (10 microM) resulted in decreased adhesion of CGTH W-2 cells to matrix substance, which was correlated with metastatic potential. Rottlerin treatment also resulted in a marked reduction in the migration of CGTH W-2 cells. Protein levels of integrin beta1, FAK, and paxillin were decreased by rottlerin. Consistent with this, immunostaining of FAK, vinculin, and paxillin revealed disassembly of the focal adhesions. Disruption of actin stress fibers was noted, which was compatible with reduced expression levels and activities of Rac-1 and Rho. The effect of rottlerin on cell migration was not attributable to inhibition of PKCdelta activity since siRNA knockdown of PKCdelta did not recapitulate the effects of rottlerin on cell adhesion and migration. Furthermore, activation of PKCdelta by phorbol esters failed to restore the rottlerin-inhibited migratory ability. The mitochondrial uncoupler, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone, was able to mimic several rottlerin's effects. In summary, we demonstrated that rottlerin inhibits the migration of CGTH W-2 cells by disassembly of focal adhesion complexes in a PKCdelta-independent manner, and might play as a mitochondrial uncoupler role in these events.
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http://dx.doi.org/10.1002/jcb.22555DOI Listing
May 2010

Macrophage activation increases the invasive properties of hepatoma cells by destabilization of the adherens junction.

FEBS Lett 2006 May 27;580(13):3042-50. Epub 2006 Apr 27.

Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, 1-1 Jen-Ai Road, Taipei 10051, Taiwan.

Tumor-associated macrophages play an important role in tumor progression, but whether they exert a tumor-progressive effect remains controversial. Here, we demonstrated that activated macrophage-conditioned medium (AMCM) obtained from RAW macrophages (RAW/AMCM) induced epithelial-mesenchymal transition (EMT) and stimulated the migratory and invasive activities of HepG2 cells, whereas control conditioned media had no effect. Epithelial-cadherin (E-cadherin) and beta-catenin staining patterns were altered at the adherens junctions by RAW/AMCM treatment, with an approximately 50% decrease in E-cadherin and beta-catenin in the cell membrane. Importantly, levels of beta-catenin-associated E-cadherin were also decreased. Following RAW/AMCM treatment, enhanced activation of c-Src was seen prior to increased tyrosine phosphorylation of beta-catenin, and this led to the destabilization of adherens junctions. Pretreatment of HepG2 cells with the Src kinase inhibitor, PP2, completely abolished the effects of RAW/AMCM on the EMT, migration, invasion, and expression and association of E-cadherin and beta-catenin. AMCMs obtained from human THP-1 monocytes and mouse peritoneal macrophages also caused disassembly of the adherens junctions and migration of HepG2 cells. Furthermore, inhibition of the epidermal growth factor receptor (EGFR) with gefitinib partially prevented the downregulation of E-cadherin and beta-catenin at the adherens junctions and migration behavior induced by RAW/AMCM. Our results suggest that activated macrophages have a tumor-progressive effect on HepG2 cells which involves the c-Src- and EGFR-dependent signaling cascades.
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http://dx.doi.org/10.1016/j.febslet.2006.04.049DOI Listing
May 2006

Pre-implantation genetic diagnosis: a preliminary report of 2 years of experience.

Chang Gung Med J 2004 Oct;27(10):726-33

Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei, Taiwan, ROC.

Background: Pre-implantation genetic diagnosis (PGD) is defined as making a diagnosis or screening embryos or gametes before implantation. It has the advantage of avoiding repeated spontaneous abortions or therapeutic termination of pregnancy resulting from abnormal embryos. Here, we present our preliminary report of 2 years of experience.

Methods: From March 2001 through October 2002, couples seeking assistance for in vitro fertilization (IVF) were referred for PGD due to chromosomal problems or for aneuploidy screening (PGD-AS). One or two blastomeres were aspirated on day 3 and analyzed using the fluorescent in situ hybridization (FISH) technique. Probes to chromosomes X, Y, and 18 were used for aneuploidy screening and individual specific probes were chosen for chromosomal translocations. Unaffected embryos were transferred on day 5.

Results: There were 25 cycles for aneuploid screening (group 1) and four cycles for chromosomal translocation (group 2). In group 1, 73 embryos were biopsied with a successful biopsy/fixation rate of 72.6% and a diagnosis rate of 96.2%. Fifteen unaffected embryos were transferred in 11 cycles, achieving two sets of twins and four singleton pregnancies (implantation rate: 53.3%). In group 2, 27 embryos were biopsied with a successful biopsy/fixation rate of 66.7% and a diagnosis rate of 88.9%. Seven non-affected embryos were transferred in three cycles, resulting in one set of twins (implantation rate: 33.3%). All antenatal amniocentesis confirmed the diagnosis. Post-natal physical examination showed no evidence of major abnormalities.

Conclusions: PGD is an alternative method for having healthy children in selected couples with chromosomal abnormalities. In addition, PGD-AS may increase the implantation rate in infertile couples seeking IVF assistance.
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October 2004