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Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes. Cell stem cell Shah, P. P., Lv, W., Rhoades, J. H., Poleshko, A., Abbey, D., Caporizzo, M. A., Linares-Saldana, R., Heffler, J. G., Sayed, N., Thomas, D., Wang, Q., Stanton, L. J., Bedi, K., Morley, M. P., Cappola, T. P., Owens, A. T., Margulies, K. B., Frank, D. B., Wu, J. C., Rader, D. J., Yang, W., Prosser, B. L., Musunuru, K., Jain, R. 2021
Abstract

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.

View details for DOI 10.1016/j.stem.2020.12.016

View details for PubMedID 33529599
Generation of Human iPSCs by Protein Reprogramming and Stimulation of TLR3 Signaling. Methods in molecular biology (Clifton, N.J.) Liu, C., Ameen, M., Himmati, S., Thomas, D., Sayed, N. 2021; 2239: 153?62
Abstract

The discovery of induced pluripotent stem cells (iPSCs) allows for establishment of human embryonic stem-like cells from various adult human somatic cells (e.g., fibroblasts), without the need for destruction of human embryos. This provides an unprecedented opportunity where patient-specific iPSCs can be subsequently differentiated to many cell types, e.g., cardiac cells and neurons, so that we can use these iPSC-derived cells to study patient-specific disease mechanisms and conduct drug testing and screening. Critically, these cells have unlimited therapeutic potentials, and there are many ongoing clinical trials to investigate the regenerative potentials of these iPSC-derivatives in humans. However, the traditional iPSC reprogramming methods have problem of insertional mutagenesis because of use of the integrating viral vectors. While a number of advances have been made to mitigate this issue, including the use of chemicals, excisable and non-integrating vectors, and use of the modified mRNA, safety remains a concern. Both integrating and non-integrating methods also suffer from many other limitations including low efficiency, variability, and tumorigenicity. The non-integrating mRNA reprogramming is of high efficiency, but it is sensitive to reagents and need approaches to reduce the immunogenic reaction. An alternative non-integrating and safer way of generating iPSCs is via direct delivery of recombinant cell-penetrating reprogramming proteins into the cells to be reprogrammed, but reprogramming efficiency of the protein-based approach is extremely low compared to the conventional virus-based nuclear reprogramming. Herein, we describe detailed steps for efficient generation of human iPSCs by protein-based reprogramming in combination with stimulation of the Toll-like receptor 3 (TLR3) innate immune pathway.

View details for DOI 10.1007/978-1-0716-1084-8_10

View details for PubMedID 33226618
In situ differentiation of human-induced pluripotent stem cells into functional cardiomyocytes on a coaxial PCL-gelatin nanofibrous scaffold. Materials science & engineering. C, Materials for biological applications Sridharan, D., Palaniappan, A., Blackstone, B. N., Dougherty, J. A., Kumar, N., Seshagiri, P. B., Sayed, N., Powell, H. M., Khan, M. 2021; 118: 111354
Abstract

Human-induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs) have been explored for cardiac regeneration and repair as well as for the development of in vitro 3D cardiac tissue models. Existing protocols for cardiac differentiation of hiPSCs utilize a 2D culture system. However, the efficiency of hiPSC differentiation to cardiomyocytes in 3D culture systems has not been extensively explored. In the present study, we investigated the efficiency of cardiac differentiation of hiPSCs to functional cardiomyocytes on 3D nanofibrous scaffolds. Coaxial polycaprolactone (PCL)-gelatin fibrous scaffolds were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. hiPSCs were cultured and differentiated into functional cardiomyocytes on the nanofibrous scaffold and compared with 2D cultures. To assess the relative efficiencies of both the systems, SEM, immunofluorescence staining and gene expression analyses were performed. Contractions of differentiated cardiomyocytes were observed in 2D cultures after 2weeks and in 3D cultures after 4weeks. SEM analysis showed no significant differences in the morphology of cells differentiated on 2D versus 3D cultures. However, gene expression data showed significantly increased expression of cardiac progenitor genes (ISL-1, SIRPA) in 3D cultures and cardiomyocytes markers (TNNT, MHC6) in 2D cultures. In contrast, immunofluorescence staining showed no substantial differences in the expression of NKX-2.5 and alpha-sarcomeric actinin. Furthermore, uniform migration and distribution of the in situ differentiated cardiomyocytes was observed in the 3D fibrous scaffold. Overall, our study demonstrates that coaxial PCL-gelatin nanofibrous scaffolds can be used as a 3D culture platform for efficient differentiation of hiPSCs to functional cardiomyocytes.

View details for DOI 10.1016/j.msec.2020.111354

View details for PubMedID 33254974
Arrhythmogenic Mechanisms in Hypokalaemia: Insights From Pre-clinical Models. Frontiers in cardiovascular medicine Tse, G., Li, K. H., Cheung, C. K., Letsas, K. P., Bhardwaj, A., Sawant, A. C., Liu, T., Yan, G., Zhang, H., Jeevaratnam, K., Sayed, N., Cheng, S. H., Wong, W. T. 2021; 8: 620539
Abstract

Potassium is the predominant intracellular cation, with its extracellular concentrations maintained between 3. 5 and 5 mM. Among the different potassium disorders, hypokalaemia is a common clinical condition that increases the risk of life-threatening ventricular arrhythmias. This review aims to consolidate pre-clinical findings on the electrophysiological mechanisms underlying hypokalaemia-induced arrhythmogenicity. Both triggers and substrates are required for the induction and maintenance of ventricular arrhythmias. Triggered activity can arise from either early afterdepolarizations (EADs) or delayed afterdepolarizations (DADs). Action potential duration (APD) prolongation can predispose to EADs, whereas intracellular Ca2+ overload can cause both EADs and DADs. Substrates on the other hand can either be static or dynamic. Static substrates include action potential triangulation, non-uniform APD prolongation, abnormal transmural repolarization gradients, reduced conduction velocity (CV), shortened effective refractory period (ERP), reduced excitation wavelength (CV * ERP) and increased critical intervals for re-excitation (APD-ERP). In contrast, dynamic substrates comprise increased amplitude of APD alternans, steeper APD restitution gradients, transient reversal of transmural repolarization gradients and impaired depolarization-repolarization coupling. The following review article will summarize the molecular mechanisms that generate these electrophysiological abnormalities and subsequent arrhythmogenesis.

View details for DOI 10.3389/fcvm.2021.620539

View details for PubMedID 33614751
The Regulation of Endothelial Function Through Hmgcr/mevalonate Pathway Mediated Yap Activity Liu, C., Liu, Y., Chen, C., Ameen, M., Yang, H., Shen, M., Rhee, J., Chen, I. Y., Sayed, N., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2020
View details for DOI 10.1161/res.127.suppl_1.325

View details for Web of Science ID 000606541500096
Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy. Science translational medicine Sayed, N., Liu, C., Ameen, M., Himmati, F., Zhang, J. Z., Khanamiri, S., Moonen, J., Wnorowski, A., Cheng, L., Rhee, J., Gaddam, S., Wang, K. C., Sallam, K., Boyd, J. H., Woo, Y. J., Rabinovitch, M., Wu, J. C. 2020; 12 (554)
Abstract

Mutations in LMNA, the gene that encodes lamin A and C, causes LMNA-related dilated cardiomyopathy (DCM) or cardiolaminopathy. LMNA is expressed in endothelial cells (ECs); however, little is known about the EC-specific phenotype of LMNA-related DCM. Here, we studied a family affected by DCM due to a frameshift variant in LMNA Human induced pluripotent stem cell (iPSC)-derived ECs were generated from patients with LMNA-related DCM and phenotypically characterized. Patients with LMNA-related DCM exhibited clinical endothelial dysfunction, and their iPSC-ECs showed decreased functionality as seen by impaired angiogenesis and nitric oxide (NO) production. Moreover, genome-edited isogenic iPSC lines recapitulated the EC disease phenotype in which LMNA-corrected iPSC-ECs showed restoration of EC function. Simultaneous profiling of chromatin accessibility and gene expression dynamics by combining assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) as well as loss-of-function studies identified Kruppel-like factor 2 (KLF2) as a potential transcription factor responsible for the EC dysfunction. Gain-of-function studies showed that treatment of LMNA iPSC-ECs with KLF2 agonists, including lovastatin, rescued the EC dysfunction. Patients with LMNA-related DCM treated with lovastatin showed improvements in clinical endothelial dysfunction as indicated by increased reactive hyperemia index. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from patients exhibiting the DCM phenotype showed improvement in CM function when cocultured with iPSC-ECs and lovastatin. These results suggest that impaired cross-talk between ECs and CMs can contribute to the pathogenesis of LMNA-related DCM, and statin may be an effective therapy for vascular dysfunction in patients with cardiolaminopathy.

View details for DOI 10.1126/scitranslmed.aax9276

View details for PubMedID 32727917
Molecular Signatures of Beneficial Class Effects of Statins on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation Tian, L., Oikonomopoulos, A., Liu, C., Kitani, T., Shrestha, R., Chen, C. L., Ong, S., Smeets, M., Karakikes, I., Sayed, N., Wu, J. C. 2020; 141 (14): 1208?10
View details for DOI 10.1161/CIRCULATIONAHA.118.035906

View details for PubMedID 32250699
Modeling Secondary Iron Overload Cardiomyopathy with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Cell reports Rhee, J. W., Yi, H., Thomas, D., Lam, C. K., Belbachir, N., Tian, L., Qin, X., Malisa, J., Lau, E., Paik, D. T., Kim, Y., Choi, B. S., Sayed, N., Sallam, K., Liao, R., Wu, J. C. 2020; 32 (2): 107886
Abstract

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

View details for DOI 10.1016/j.celrep.2020.107886

View details for PubMedID 32668256
HIF1? Regulates Early Metabolic Changes due to Activation of Innate Immunity in Nuclear Reprogramming. Stem cell reports Liu, C., Ruan, H., Himmati, F., Zhao, M. T., Chen, C. C., Makar, M., Chen, I. Y., Sallam, K., Mocarski, E. S., Sayed, D., Sayed, N. 2020; 14 (2): 192?200
Abstract

Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1? (HIF1?), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1? is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1? regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.

View details for DOI 10.1016/j.stemcr.2020.01.006

View details for PubMedID 32048999
Effects of Spaceflight on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function. Stem cell reports Wnorowski, A., Sharma, A., Chen, H., Wu, H., Shao, N., Sayed, N., Liu, C., Countryman, S., Stodieck, L. S., Rubins, K. H., Wu, S. M., Lee, P. H., Wu, J. C. 2019
Abstract

With extended stays aboard the International Space Station (ISS) becoming commonplace, there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5weeks and their gene expression, structure, and functions were compared with ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated that 2,635 genes were differentially expressed among flight, post-flight, and ground control samples, including genes involved in mitochondrial metabolism. This study represents the first use of hiPSC technology to model the effects of spaceflight on human cardiomyocyte structure and function.

View details for DOI 10.1016/j.stemcr.2019.10.006

View details for PubMedID 31708475
Vismione B Interferes with Trypanosoma cruzi Infection of Vero Cells and Human Stem Cell-Derived Cardiomyocytes. The American journal of tropical medicine and hygiene Sass, G., Tsamo, A. T., Chounda, G. A., Nangmo, P. K., Sayed, N., Bozzi, A., Wu, J. C., Nkengfack, A. E., Stevens, D. A. 2019
Abstract

Traditional African medicine is a source of new molecules that might be useful in modern therapeutics. We tested ten limonoids, six quinones, one xanthone, one alkaloid, and one cycloartane, isolated from four Cameroonian medicinal plants, and one plant-associated endophytic fungus, against Trypanosoma cruzi, the etiological agent of Chagas disease (CD). Vero cells, or human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (hiPSC-CM) were infected with T. cruzi trypomastigotes (discrete typing unit types I or II). Infection took place in the presence of drugs, or 24 hours before drug treatment. Forty-eight hours after infection, infection rates and parasite multiplication were evaluated by Giemsa stain. Cell metabolism was measured to determine functional integrity. In Vero cells, several individual molecules significantly affected T. cruzi infection and multiplication with no, or minor, effects on cell viability. Reduced infection rates and multiplication by the quinone vismione B was superior to the commonly used therapeutic benznidazole (BNZ). The vismione B concentration inhibiting 50% of T. cruzi infection (IC50) was 1.3 M. When drug was applied after infection, anti-Trypanosoma effects of vismione B [10 M) were significantly stronger than effects of BNZ (23 M). Furthermore, in hiPSC-CM cultures, infection and multiplication rates in the presence of vismione B (10 M) were significantly lower than in BNZ (11.5 M), without showing signs of cytotoxicity. Our data indicate that vismione B is more potent against T. cruzi infection and multiplication than BNZ, with stronger effects on established infection. Vismione B, therefore, might become a promising lead molecule for treatment development for CD.

View details for DOI 10.4269/ajtmh.19-0350

View details for PubMedID 31571568
Adiponectin Receptor 3 is Associated With Endothelial Nitric Oxide Synthase Dysfunction and Predicts Insulin Resistance in South Asians Chandy, M., Sayed, N., Lau, E., Liu, C., Wei Tzu-Tang, Chen, I. Y., Thomas, D., Rhee, J., Oh, B., Pepic, L., Husain, M., Quertermous, T., Nallamshetty, S., Wu, J. LIPPINCOTT WILLIAMS & WILKINS. 2019
View details for Web of Science ID 000511467800216
Studying Cardiovascular Effects of Marijuana on Healthy Individuals Using Human Derived Induced Pluripotent Stem Cells Wei, T., Chandy, M., Chen, I. Y., Wo, H., Khanamiri, S., Nishiga, M., Seidl, F., Sayed, N., Liu, C., Rhee, J., Obal, D., Chour, T., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2019
View details for Web of Science ID 000511467800293
Human-induced Pluripotent Stem Cell-derived Cardiomyocytes as a Model for Trastuzumab-Induced Cardiac Dysfunction Kitani, T., Ong, S., Lam, C. K., Rhee, J., Zhang, J. Z., Oikonomopoulos, A., Ma, N., Tian, L., Lee, J., Telli, M. L., Witteles, R. M., Sharma, A., Sayed, N., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2019
View details for Web of Science ID 000511467800463
A Combination of Itraconazole and Amiodarone Is Highly Effective against Trypanosoma cruzi Infection of Human Stem Cell-Derived Cardiomyocytes. The American journal of tropical medicine and hygiene Sass, G., Madigan, R. T., Joubert, L., Bozzi, A., Sayed, N., Wu, J. C., Stevens, D. A. 2019
Abstract

Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other non-endemic countries, rising case numbers have also been observed. The currently used drugs are benznidazole (BNZ) and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole (ICZ), originally an antifungal, in combination with amiodarone (AMD), an antiarrhythmic, with the goal of interfering with T. cruzi infection. Human pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPSC-CMs). Vero cells or hiPSC-CMs were infected with T. cruzi trypomastigotes of the II or I strain in the presence of ICZ and/or AMD. After 48 hours, cells were Giemsa stained, and infection and multiplication were evalutated microscopically. Trypanosoma cruzi infection and multiplication were evalutated also by electron microscopy. BNZ was used as a reference compound. Cell viability in the presence of test substances was assessed. Itraconazole and AMD showed strain- and dose-dependent interference with T. cruzi infection and multiplication in Vero cells or hiPSC-CMs. Combinations of ICZ and AMD were more effective against T. cruzi than the single substances, or BNZ, without affecting host cell metabolism, and better preserving host cell integrity during infection. Our in vitro data in hiPSC-CMs suggest that a combination of ICZ and AMD might serve as a treatment option for CD in patients, but that different responses due to T. cruzi strain differences have to be taken into account.

View details for DOI 10.4269/ajtmh.19-0023

View details for PubMedID 31219005
Cancer therapy-induced cardiomyopathy: can human induced pluripotent stem cell modelling help prevent it? EUROPEAN HEART JOURNAL Stack, J. P., Moslehi, J., Sayed, N., Wu, J. C. 2019; 40 (22): 1764?70
View details for DOI 10.1093/eurheartj/ehx811

View details for Web of Science ID 000490131200007
Human-Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Patients With Breast Cancer CIRCULATION Kitani, T., Ong, S., Lam, C., Rhee, J., Zhang, J. Z., Oikonomopoulos, A., Ma, N., Tian, L., Lee, J., Telli, M. L., Witteles, R. M., Sharma, A., Sayed, N., Wu, J. C. 2019; 139 (21): 2451?65
View details for DOI 10.1161/CIRCULATIONAHA.118.037357

View details for Web of Science ID 000469018300014
A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles CELL STEM CELL Zhang, J. Z., Termglinchan, V., Shao, N., Itzhaki, I., Liu, C., Ma, N., Tian, L., Wang, V. Y., Chang, A. Y., Guo, H., Kitani, T., Wu, H., Lam, C., Kodo, K., Sayed, N., Blau, H. M., Wu, J. C. 2019; 24 (5): 802-+
View details for DOI 10.1016/j.stem.2019.02.015

View details for Web of Science ID 000466726500017
Personalized medicine in cardio-oncology: the role of induced pluripotent stem cell CARDIOVASCULAR RESEARCH Sayed, N., Ameen, M., Wu, J. C. 2019; 115 (5): 949?59
View details for DOI 10.1093/cvr/cvz024

View details for Web of Science ID 000466849100014
Glucocorticoid Receptor-Binding and Transcriptome Signature in Cardiomyocytes JOURNAL OF THE AMERICAN HEART ASSOCIATION Severinova, E., Alikunju, S., Deng, W., Dhawan, P., Sayed, N., Sayed, D. 2019; 8 (6)
View details for DOI 10.1161/JAHA.118.011484

View details for Web of Science ID 000484573500028
Human Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Breast Cancer Patients. Circulation Kitani, T., Ong, S. G., Lam, C. K., Rhee, J. W., Zhang, J. Z., Oikonomopoulos, A., Ma, N., Tian, L., Lee, J., Telli, M. L., Witteles, R. M., Sharma, A., Sayed, N., Wu, J. C. 2019
Abstract

Molecular targeted chemotherapies have been shown to significantly improve cancer patient outcomes, but often cause cardiovascular side effects that limit their use and impair patients' quality of life. Cardiac dysfunction induced by these therapies, especially trastuzumab, shows a distinct cardiotoxic clinical phenotype compared to cardiotoxicity induced by conventional chemotherapies.We employed the human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform to determine the underlying cellular mechanisms in trastuzumab-induced cardiac dysfunction. We assessed the effects of trastuzumab on structural and functional properties in iPSC-CMs from healthy individuals and performed RNA-sequencing (RNA-seq) to further examine the effect of trastuzumab on iPSC-CMs. We also generated iPSCs from patients receiving trastuzumab and examined whether patients' phenotype could be recapitulated in vitro using patient-specific iPSC-CMs.We found that clinically relevant doses of trastuzumab significantly impaired the contractile and calcium handling properties of iPSC-CMs without inducing cardiomyocyte death or sarcomeric disorganization. RNA-seq and subsequent functional analysis revealed mitochondrial dysfunction and altered cardiac energy metabolism pathway as primary causes of trastuzumab-induced cardiotoxic phenotype. Human iPSC-CMs generated from patients who received trastuzumab and experienced severe cardiac dysfunction were more vulnerable to trastuzumab treatment, compared to iPSC-CMs generated from patients who did not experience cardiac dysfunction following trastuzumab therapy. Importantly, metabolic modulation with AMPK activators could avert the adverse effects induced by trastuzumab.Our results indicate that alterations in cellular metabolic pathways in cardiomyocytes could be a key mechanism underlying the development of cardiac dysfunction following trastuzumab therapy; therefore, targeting the altered metabolism may be a promising therapeutic approach for trastuzumab-induced cardiac dysfunction.

View details for PubMedID 30866650
Glucocorticoid Receptor-Binding and Transcriptome Signature in Cardiomyocytes. Journal of the American Heart Association Severinova, E., Alikunju, S., Deng, W., Dhawan, P., Sayed, N., Sayed, D. 2019; 8 (6): e011484
Abstract

Background An increase in serum cortisol has been identified as a risk factor for cardiac failure, which highlights the impact of glucocorticoid signaling in cardiomyocytes and its influence in the progression of failure. Dexamethasone, a synthetic glucocorticoid, is sufficient for induction of cardiomyocyte hypertrophy, but little is known of the glucocorticoid receptor (GR) genome-binding and -dependent transcriptional changes that mediate this phenotype. Methods and Results In this study using high-resolution sequencing, we identified genomic targets of GR and associated change in the transcriptome after 1 and 24�hours of dexamethasone treatment. We showed that GR associates with 6482 genes in the cardiac genome, with differential regulation of 738 genes. Interestingly, alignment of the chromatin immunoprecipitation and RNA sequencing data show that, after 1�hour, 69% of differentially regulated genes are associated with GR and identify as regulators of RNA pol II-dependent transcription. Conversely, after 24�hours only 45% of regulated genes are associated with GR and involved in dilated and hypertrophic cardiomyopathies as well as other growth-related pathways. In addition, our data also reveal that a majority of genes (76.42%) associated with GR show incremental changes in transcript abundance and are genes involved in basic cellular processes that might be regulated by the dynamics of promoter-paused RNA pol II, as seen in hearts undergoing hypertrophy. In vivo administration of dexamethasone resulted in similar changes in the cardiac transcriptome, as seen in isolated cardiomyocytes. Conclusions Our data reveal genome-wide GR binding sites in cardiomyocytes, identify novel targets and GR-dependent change in the transcriptome that induces and contributes to cardiomyocyte hypertrophy.

View details for PubMedID 30866692
Marked Vascular Dysfunction in a Case of Peripartum Cardiomyopathy. Journal of vascular research Khanamiri, S., Rhee, J. W., Paik, D. T., Chen, I. Y., Liu, C., Sayed, N. 2019; 56 (1): 11?15
Abstract

Peripartum cardiomyopathy (PPCM) is a rare form of congestive heart failure characterized by left ventricular dysfunction that develops towards the end of pregnancy or during the early postpartum phase. Even though the majority of PPCM patients show partial or complete recovery of their heart functions, the mortality rate of PPCM remains high. Previous research has suggested that vascular dysfunction triggered by late-gestational hormones and potent anti-angiogenic factors play key roles in the pathogenesis of PPCM; however, the exact mechanisms remain elusive due to limited patient tissues for characterization. Here, we report a case of PPCM where the coronary vessels from the patient's explanted heart showed marked vascular dysfunction with impaired nitric oxide response. Importantly, these vessels exhibited deficient adenosine-mediated vasorelaxation when subjected to myograph studies, suggesting impaired Kv7 ion channels. Results from this work may lead to new therapeutic strategies for improving Kv7 function in PPCM patients.

View details for PubMedID 30763932
An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium. ACS nano Yang, H., Qin, X., Wang, H., Zhao, X., Liu, Y., Wo, H. T., Liu, C., Nishiga, M., Chen, H., Ge, J., Sayed, N., Abilez, O. J., Ding, D., Heilshorn, S. C., Li, K. 2019
Abstract

A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

View details for DOI 10.1021/acsnano.9b03343

View details for PubMedID 31149806
Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as�a�Model to Study Trypanosoma cruzi Infection. Stem cell reports Bozzi, A., Sayed, N., Matsa, E., Sass, G., Neofytou, E., Clemons, K. V., Correa-Oliveira, R., Stevens, D. A., Wu, J. C. 2019
Abstract

Chagas disease (ChD) is one of the most neglected tropical diseases, with cardiomyopathy being the main cause of death in Trypanosoma cruzi-infected patients. As the parasite actively replicates in cardiomyocytes (CMs), the heart remains a key target organ in the pathogenesis of ChD. Here we modeled ChD using human induced pluripotent stem cell-derived CMs (iPSC-CMs) to understand the complex interplay between the parasite and host cells. We showed that iPSC-CMs can get infected with the T. cruzi Y strain and that all parasite cycle stages can be identified in our model system. Importantly, characterization of T. cruzi-infected iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, these infected iPSC-CMs exhibited a pro-inflammatory profile as indicated by significantly elevated cytokine levels�and cell-trafficking regulators. We believe our iPSC-CM model is a valuable platform to explore new treatment strategies for ChD.

View details for DOI 10.1016/j.stemcr.2019.04.017

View details for PubMedID 31105048
Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells. Circulation research Paik, D. T., Tian, L., Lee, J., Sayed, N., Chen, I. Y., Rhee, S., Rhee, J., Kim, Y., Wirka, R. C., Buikema, J. W., Wu, S. M., Red-Horse, K., Quertermous, T., Wu, J. C. 2018
Abstract

Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. Conclusions: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

View details for PubMedID 29986945
Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells. Circulation Ma, N., Zhang, J., Itzhaki, I., Zhang, S. L., Chen, H., Haddad, F., Kitani, T., Wilson, K. D., Tian, L., Shrestha, R., Wu, H., Lam, C. K., Sayed, N., Wu, J. C. 2018
Abstract

Background -The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. Methods -To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Humaninduced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUSMYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). Results -The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. Conclusions -Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrierspecific manner in a dish. As such, this platform represents a promising VUS riskassessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.

View details for PubMedID 29914921
REVERSING LMNA MUTATION PHENOTYPE IN CARDIOMYOCYTES WITH OLMESARTAN: CONNECTING MONOGENIC DISEASE TO DRUGS VIA TRANSCRIPTIONAL SIGNATURES Kort, E., Sayed, N., Wu, J., Jovinge, S. ELSEVIER SCIENCE INC. 2018: 897
View details for DOI 10.1016/S0735-1097(18)31438-4

View details for Web of Science ID 000429659702147
IMMUNE PROFILE OF HEALTHY CARDIOVASCULAR AGING: INSIGHTS FROM A POPULATION-BASED STUDY AND NETWORK MODELING Bouajila, S., Moneghetti, K., Kobayashi, Y., Gomari, F. A., Abbasi, F., Davis, M. M., Wu, J. C., Kuznetsova, T., Sayed, N., Haddad, F. ELSEVIER SCIENCE INC. 2018: 1657
View details for Web of Science ID 000429659703307
Cancer therapy-induced cardiomyopathy: can human induced pluripotent stem cell modelling help prevent it? European heart journal Stack, J. P., Moslehi, J., Sayed, N., Wu, J. C. 2018
Abstract

Cardiotoxic effects from cancer therapy are a major cause of morbidity during cancer treatment. Unexpected toxicity can occur during treatment and/or after completion of therapy, into the time of cancer survivorship. While older drugs such as anthracyclines have well-known cardiotoxic effects, newer drugs such as tyrosine kinase inhibitors, proteasome inhibitors, and immunotherapies also can cause diverse cardiovascular and metabolic complications. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly being used as instruments for disease modelling, drug discovery, and mechanistic toxicity studies. Promising results with hiPSC-CM chemotherapy studies are raising hopes for improving cancer therapies through personalized medicine and safer drug development. Here, we review the cardiotoxicity profiles of common chemotherapeutic agents as well as efforts to model them in vitro using hiPSC-CMs.

View details for PubMedID 29377985
Big bottlenecks in cardiovascular tissue engineering. Communications biology Huang, N. F., Serpooshan, V., Morris, V. B., Sayed, N., Pardon, G., Abilez, O. J., Nakayama, K. H., Pruitt, B. L., Wu, S. M., Yoon, Y., Zhang, J., Wu, J. C. 2018; 1: 199
Abstract

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges.

View details for PubMedID 30480100
Generation of Endothelial Cells from Human Induced Pluripotent Stem Cells. Bio-protocol Bio101 Liu, C., Linling, C., Chen, C. L., Sayed, N. 2018
View details for DOI 10.21769/BioProtoc.3086
Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond. Development (Cambridge, England) Liu, C., Oikonomopoulos, A., Sayed, N., Wu, J. C. 2018; 145 (5)
Abstract

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimicin vivotissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling.

View details for PubMedID 29519889
Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming STEM CELLS Sayed, N., Ospino, F., Himmati, F., Lee, J., Chanda, P., Mocarski, E. S., Cooke, J. P. 2017; 35 (5): 1197-1207
Abstract

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/stem.2607

View details for Web of Science ID 000400017200008

View details for PubMedID 28276156
Towards Cardio-Precision medicine EUROPEAN HEART JOURNAL Sayed, N., Wu, J. C. 2017; 38 (14): 1014?16
View details for DOI 10.1093/eurheartj/ehx089

View details for Web of Science ID 000398576800004
High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells. Science translational medicine Sharma, A., Burridge, P. W., McKeithan, W. L., Serrano, R., Shukla, P., Sayed, N., Churko, J. M., Kitani, T., Wu, H., Holmstr�m, A., Matsa, E., Zhang, Y., Kumar, A., Fan, A. C., Del �lamo, J. C., Wu, S. M., Moslehi, J. J., Mercola, M., Wu, J. C. 2017; 9 (377)
Abstract

Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

View details for DOI 10.1126/scitranslmed.aaf2584

View details for PubMedID 28202772
Paying the Toll in Nuclear Reprogramming. Frontiers in cell and developmental biology Liu, C., Himmati, F., Sayed, N. 2017; 5: 70
Abstract

The ability to reverse lineage-committed cells toward pluripotent stem cells or to another cell type is one of the ultimate goals in regenerative medicine. We recently discovered that activation of innate immunity, through Toll-like receptor 3, is required during this conversion of cell fate by causing global changes in the expression and activity of epigenetic modifiers. Here we discuss, in a comprehensive manner, the recent studies on the role of innate immunity in nuclear reprogramming and transdifferentiation, the underlying mechanisms, and its role in regenerative medicine.

View details for PubMedID 28861413
Getting to the Heart of the Matter: A Perspective on Cardiomyocyte Biology Annals of Vascular Medicine & Research Chen, F. M., Tse, G., Ma, S., Sayed, N., Wong, W. T. 2017; 4 (4): 1067
Molecular and functional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs. Proceedings of the National Academy of Sciences of the United States of America Zhao, M. T., Chen, H., Liu, Q., Shao, N. Y., Sayed, N., Wo, H. T., Zhang, J. Z., Ong, S. G., Liu, C., Kim, Y., Yang, H., Chour, T., Ma, H., Gutierrez, N. M., Karakikes, I., Mitalipov, S., Snyder, M. P., Wu, J. C. 2017
Abstract

Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.

View details for PubMedID 29203658
Transcriptome Profiling of Patient-Specific Human iPSC-Cardiomyocytes Predicts Individual Drug Safety and Efficacy Responses In�Vitro. Cell stem cell Matsa, E., Burridge, P. W., Yu, K., Ahrens, J. H., Termglinchan, V., Wu, H., Liu, C., Shukla, P., Sayed, N., Churko, J. M., Shao, N., Woo, N. A., Chao, A. S., Gold, J. D., Karakikes, I., Snyder, M. P., Wu, J. C. 2016; 19 (3): 311-325
Abstract

Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient�variation than intra-patient variation, verifying�that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in�vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.

View details for DOI 10.1016/j.stem.2016.07.006

View details for PubMedID 27545504
Translation of Human-Induced Pluripotent Stem Cells From Clinical Trial in a Dish to Precision Medicine JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Sayed, N., Liu, C., Wu, J. C. 2016; 67 (18): 2161-2176
Abstract

The prospect of changing the plasticity of terminally differentiated cells toward pluripotency has completely altered the outlook for biomedical research. Human-induced pluripotent stem cells (iPSCs) provide a new source of therapeutic cells�free from the ethical issues or immune barriers of human embryonic stem cells. iPSCs also confer considerable advantages over conventional methods of studying human diseases. Since its advent, iPSC technology has expanded with 3 major applications: disease modeling, regenerative therapy, and drug discovery. Here we discuss, in a comprehensive manner, the recent advances in iPSC technology in relation to basic, clinical, and population health.

View details for DOI 10.1016/j.jacc.2016.01.083

View details for Web of Science ID 000375406100011

View details for PubMedID 27151349
Vascular Aging: Implications for Cardiovascular Disease and Therapy. Translational medicine (Sunnyvale, Calif.) Ghebre, Y. T., Yakubov, E., Wong, W. T., Krishnamurthy, P., Sayed, N., Sikora, A. G., Bonnen, M. D. 2016; 6 (4)
Abstract

The incidence and prevalence of cardiovascular disease is highest among the elderly, in part, due to deleterious effects of advancing age on the heart and blood vessels. Aging, a known cardiovascular risk factor, is progressively associated with structural and functional changes to the vasculature including hemodynamic disturbance due to increased oxidative stress, premature cellular senescence and impairments in synthesis and/or secretion of endothelium-derived vasoactive molecules. These molecular and physiological changes lead to vessel wall stiffening and thickening, as well as other vascular complications that culminate to loss of vascular tone regulation and endothelial function. Intriguingly, the vessel wall, a biochemically active structure composed of collagen, connective tissue, smooth muscle and endothelial cells, is adversely affected by processes involved in premature or normal aging. Notably, the inner most layer of the vessel wall, the endothelium, becomes senescent and dysfunctional with advancing age. As a result, its ability to release vasoactive molecules such as acetylcholine (ACh), prostacyclin (PGI2), endothelium-derived hyperpolarizing factor (EDHF), and nitric oxide (NO) is reduced and the cellular response to these molecules is also impaired. By contrast, the vascular endothelium increases its generation and release of reactive oxygen (ROS) and nitrogen (RNS) species, vasoconstrictors such as endothelin (ET) and angiotensin (AT), and endogenous inhibitors of NO synthases (NOSs) to block NO. This skews the balance of the endothelium in favor of the release of highly tissue reactive and harmful molecules that promote DNA damage, telomere erosion, senescence, as well as stiffened and hardened vessel wall that is prone to the development of hypertension, diabetes, atherosclerosis and other cardiovascular risk factors. This Review discusses the impact of advancing age on cardiovascular health, and highlights the cellular and molecular mechanisms that underlie age-associated vascular changes. In addition, the role of pharmacological interventions in preventing or delaying age-related cardiovascular disease is discussed.

View details for PubMedID 28932625
Vascular Aging: Implications for Cardiovascular Disease and Therapy Translational Medicine Ghebre, Y. T., Yakubov, E., Wong, W., Krishnamurthy, P., Sayed, N., Sikora, A. G., Bonnen, M. D., et al 2016
View details for DOI 10.4172/2161-1025.1000183
Response to Letter Regarding Article "Transdifferentiation of Human Fibroblasts to Endothelial Cells: Role of Innate Immunity" CIRCULATION Cooke, J. P., Meng, S., Wong, W. T., Sayed, N., Ospino, F., Lee, J., Jha, A., Dexheimer, P., Aronow, B. 2015; 132 (15): E197-E197
View details for DOI 10.1161/CIRCULATIONAHA.115.016792

View details for Web of Science ID 000362563200008

View details for PubMedID 26459087
Transdifferentiation of human fibroblasts to endothelial cells: role of innate immunity. Circulation Sayed, N., Wong, W. T., Ospino, F., Meng, S., Lee, J., Jha, A., Dexheimer, P., Aronow, B. J., Cooke, J. P. 2015; 131 (3): 300-309
Abstract

-Cell fate is fluid, and may be altered experimentally by the forced expression of master regulators mediating cell lineage. Such reprogramming has been achieved using viral vectors encoding transcription factors. We recently discovered that the viral vectors are more than passive vehicles for transcription factors, as they participate actively in the process of nuclear reprogramming to pluripotency by increasing epigenetic plasticity. Based on this recognition, we hypothesized that small molecule activators of toll-like receptor 3 (TLR3), together with external microenvironmental cues that drive EC specification, might be sufficient to induce transdifferentiation of fibroblasts into ECs (iECs).-We show that TLR3 agonist Poly I:C, combined with exogenous EC growth factors, transdifferentiated human fibroblasts into ECs. These iECs were comparable to HMVEC in immunohistochemical, genetic and functional assays, including the ability to form capillary-like structures and to incorporate acetylated-LDL. Furthermore, iECs significantly improved limb perfusion and neovascularization in the murine ischemic hindlimb. Finally, using genetic knockdown studies, we find that the effective transdifferentiation of human fibroblasts to endothelial cells requires innate immune activation.-This study suggests that manipulation of innate immune signaling may be generally used to modify cell fate. As similar signaling pathways are activated by damage associated molecular patterns, epigenetic plasticity induced by innate immunity may play a fundamental role in transdifferentiation during wound healing and regeneration. Finally, this study is a first step toward development of a small molecule strategy for therapeutic transdifferentiation for vascular disease.

View details for DOI 10.1161/CIRCULATIONAHA.113.007394

View details for PubMedID 25359165
Innate immunity and epigenetic plasticity in cellular reprogramming CURRENT OPINION IN GENETICS & DEVELOPMENT Cooke, J. P., Sayed, N., Lee, J., Wong, W. T. 2014; 28: 89-91
Abstract

Somatic cells can be reprogrammed to express the features of pluripotent cells, in that they can be differentiated into all three germ layers, and that they have the ability to replicate indefinitely. Recent studies suggest that the efficient induction of pluripotency requires the activation of innate immunity.

View details for DOI 10.1016/j.gde.2014.11.002

View details for Web of Science ID 000347764300015

View details for PubMedID 25461456

View details for PubMedCentralID PMC4262715
Therapeutic transdifferentiation: can we generate cardiac tissue rather than scar after myocardial injury? Methodist DeBakey cardiovascular journal Sayed, N., Wong, W. T., Cooke, J. P. 2013; 9 (4): 210-212
Abstract

After myocardial injury, the cardiac muscle does not regenerate and heals by forming a scar. This process results in loss of heart function and ultimately heart failure. Recent application of reprogramming technology, where forced expression of master regulators convert scar-forming cells to become cardiovascular cells in vivo, has fueled new hope for the development of therapies targeting heart disease.

View details for PubMedID 24298312
Induced pluripotent stem cells: how they will change the practice of cardiovascular medicine. Methodist DeBakey cardiovascular journal Wong, W. T., Sayed, N., Cooke, J. P. 2013; 9 (4): 206-209
Abstract

Induced pluripotent stem cells (iPSCs) can be generated from adult somatic tissues by the forced expression of a few defined transcription factors, including Oct4, Sox2, Klf4, and c-Myc. iPSC technology holds tremendous promises for therapeutic cardiovascular regeneration because of the cells' unlimited capacity for proliferation and differentiation into all cell lineages. The iPSCs can be generated from somatic cells of patients with a genetic basis for their disease so as to understand the pathobiology of the disorder. This disease modeling can be adapted to high-throughput screens to discover new therapeutic molecules. Finally, the iPSC technology may enable personalized cell therapies, while avoiding the ethical concerns surrounding human embryonic stem cells. Intensive efforts are underway to develop reliable methods to guide stem cell differentiation into cardiovascular lineages in the treatment of peripheral artery disease and heart diseases. Studies of disease pathogenesis and drug discovery using iPSC technology shall advance the discovery of novel treatments for cardiovascular diseases.

View details for PubMedID 24298311
Hypothalamic S-Nitrosylation Contributes to the Counter-Regulatory Response Impairment following Recurrent Hypoglycemia PLOS ONE Fioramonti, X., Deak, A., Deshpande, S., Carneiro, L., Zhou, C., Sayed, N., Orban, B., Berlin, J. R., Penicaud, L., Leloup, C., Beuve, A., Routh, V. H. 2013; 8 (7)
Abstract

Hypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation.Acute insulin-hypoglycemia increased VMH ROS levels by 49�6.3%. RH increased VMH sGC S-nitrosylation. Increasing VMH S-nitrosylation with intracerebroventricular injection of the nitrosylating agent S-nitroso-L-cysteine (CSNO) was associated with decreased glucagon secretion during hypoglycemic clamp. Finally, in RH rats pre-treated with NAC (0.5% in drinking water for 9 days) hypoglycemia-induced VMH ROS production was prevented and glucagon and epinephrine production was not blunted in response to subsequent insulin-hypoglycemia.These data suggest that NAC may be clinically useful in preventing impaired CRR in patients undergoing intensive-insulin therapy.

View details for DOI 10.1371/journal.pone.0068709

View details for Web of Science ID 000322391400021

View details for PubMedID 23894333
Leveraging the innate immunity pathway for transdifferentiation of fibroblasts to endothelial cells Sayed, N., Wong, W. T., Cooke, J. P. SAGE PUBLICATIONS LTD. 2013: 153?54
View details for Web of Science ID 000319606800011
Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming CELL Lee, J., Sayed, N., Hunter, A., Au, K. F., Wong, W. H., Mocarski, E. S., Pera, R. R., Yakubov, E., Cooke, J. P. 2012; 151 (3): 547-558
Abstract

Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripotent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved exceedingly inefficient. We discovered a striking difference in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogramming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain- and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables efficient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory pathways are required for efficient nuclear reprogramming in the induction of pluripotency.

View details for DOI 10.1016/j.cell.2012.09.034

View details for PubMedID 23101625
Endothelial Cells Derived From Nuclear Reprogramming CIRCULATION RESEARCH Wong, W. T., Huang, N. F., Botham, C. M., Sayed, N., Cooke, J. P. 2012; 111 (10): 1363-1375
Abstract

The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health, the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell (EC) culture have been developed and refined. Recently, methods to derive ECs from pluripotent cells have extended the scientific range of human EC studies. Pluripotent stem cells may be generated, expanded, and then differentiated into ECs for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific ECs to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of ECs from stem cells; their characterization by genetic, histological, and functional studies; and their translational applications.

View details for DOI 10.1161/CIRCRESAHA.111.247213

View details for Web of Science ID 000310501300017

View details for PubMedID 23104878

View details for PubMedCentralID PMC3526979
Toll-Like Receptor 3 Activation Promotes Efficient Nuclear Reprogramming and Endothelial Differentiation Basic Cardiovascular Sciences Scientific Session Sayed, N., Lee, J., Hunter, A., Au, K. F., Wong, W., Mocarski, E., Pera, R. R., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2012
View details for Web of Science ID 000312506400344
NaHS relaxes rat cerebral artery in vitro via inhibition of L-type voltage-sensitive Ca2+ channel PHARMACOLOGICAL RESEARCH Tian, X. Y., Wong, W. T., Sayed, N., Luo, J., Tsang, S. Y., Bian, Z. X., Lu, Y., Cheang, W. S., Yao, X., Chen, Z. Y., Huang, Y. 2012; 65 (2): 239-246
Abstract

H(2)S, a gaseous signalling molecule, relaxes blood vessels partly through activation of ATP-sensitive K(+) channels. It is however unclear whether H(2)S or its donors could affect other ion transporting proteins. The present study examined the hypothesis that NaHS, a H(2)S donor inhibits voltage-sensitive Ca(2+) channels and thus relaxes vascular smooth muscle cells (VSMC) in the cerebral arteries. NaHS dilated cerebral arteries from Sprague-Dawley rats with the same potency against pre-contraction by 5-HT and 60 mmol/L KCl, which were unaffected by several K(+) channel blockers, N(G)-nitro-l-arginine methyl ester or indomethacin, as assessed in wire myograph under an isometric condition. Likewise, NaHS also dilated cerebral arteries against myogenic constriction in pressurized myograph under an isobaric condition. NaHS concentration-dependently inhibited CaCl(2)-induced contraction in Ca(2+)-free, 60mM K(+)-containing Krebs solution. Patch clamp recordings showed that NaHS reduced the amplitude of l-type Ca(2+) currents in single myocytes isolated enzymatically from the cerebral artery. Calcium fluorescent imaging using fluo-4 showed a reduced [Ca(2+)](i) in 60 mmol/L KCl-stimulated rat cerebral arteries in response to NaHS. H(2)S precursor l-cysteine-induced relaxation in cerebral arteries was inhibited by cystathionine ?-lyase (CSE) inhibitor dl-propargylglycine. CSE was expressed in cerebral arteries. In summary, NaHS dilates rat cerebral arteries by reducing l-type Ca(2+) currents and suppressing [Ca(2+)](i) of arterial myocyte, indicating that NaHS relaxes cerebral arteries primarily through inhibiting Ca(2+) influx via Ca(2+) channels.

View details for DOI 10.1016/j.phrs.2011.11.006

View details for Web of Science ID 000301868300012

View details for PubMedID 22133671
Protein kinase G phosphorylates soluble guanylyl cyclase on serine 64 and inhibits its activity ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Zhou, Z., Sayed, N., Pyriochou, A., Roussos, C., Fulton, D., Beuve, A., Papapetropoulos, A. 2008; 28 (10): 1803-1810
Abstract

Binding of nitric oxide (NO) to soluble guanylyl cyclase (sGC) leads to increased cGMP synthesis that activates cGMP-dependent protein kinase (PKG). Herein, we tested whether sGC activity is regulated by PKG.Overexpression of a constitutively active form of PKG (DeltaPKG) stimulated (32)P incorporation into the alpha1 subunit. Serine to alanine mutation of putative sites revealed that Ser64 is the main phosphorylation site for PKG. Using a phospho-specific antibody we observed that endogenous sGC phosphorylation on Ser 64 increases in cells and tissues exposed to NO, in a PKG-inhibitable manner. Wild-type (wt) sGC coexpressed with DeltaPKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A alpha1/beta1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D alpha1 phosphomimetic /beta1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D alpha1/beta1 compared to wild-type sGC. Expression of a phosphorylation-deficient sGC showed enhanced responsiveness to endothelium-derived NO, reduced desensitization to acute NO exposure, and allowed for greater VASP phosphorylation.We conclude that PKG phosphorylates sGC on Ser64 of the alpha1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop.

View details for DOI 10.1161/ATVBAHA.108.165043

View details for Web of Science ID 000259278200020

View details for PubMedID 18635821
Nitroglycerin-induced S-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance CIRCULATION RESEARCH Sayed, N., Kim, D. D., Fioramonti, X., Iwahashi, T., Duran, W. N., Beuve, A. 2008; 103 (6): 606-614
Abstract

Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy. We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation.

View details for DOI 10.1161/CIRCRESAHA.108.175133

View details for Web of Science ID 000259252500011

View details for PubMedID 18669924
PAS-mediated dimerization of soluble guanylyl cyclase revealed by signal transduction histidine kinase domain crystal structure JOURNAL OF BIOLOGICAL CHEMISTRY Ma, X., Sayed, N., Baskaran, P., Beuve, A., van den Akker, F. 2008; 283 (2): 1167-1178
Abstract

Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) because it is often associated with the heme-nitric oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 A resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCbeta1 H-NOXA homodimerization, heterodimerization of full-length heterodimeric sGC was mostly unaffected, likely due to the additional dimerization contacts of sGC in the coiled-coil and catalytic domains. Exceptions are mutations sGCalpha1 F285A and sGCbeta1 F217A, which each caused a drastic drop in NO stimulated activity, and mutations sGCalpha1 Q368A and sGCbeta1 Q309A, which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture.

View details for DOI 10.1074/jbc.M706218200

View details for Web of Science ID 000252128100057

View details for PubMedID 18006497
Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sayed, N., Baskaran, P., Ma, X., van den Akker, F., Beuve, A. 2007; 104 (30): 12312-12317
Abstract

The molecular mechanism of desensitization of soluble guanylyl cyclase (sGC), the NO receptor, has long remained unresolved. Posttranslational modification and redox state have been postulated to affect sGC sensitivity to NO but evidence has been lacking. We now show that sGC can be S-nitrosylated in primary aortic smooth muscle cells by S-nitrosocysteine (CSNO), an S-nitrosylating agent, in human umbilical vein endothelial cells after vascular endothelial growth factor treatment and in isolated aorta after sustained exposure to acetylcholine. Importantly, we show that S-nitrosylation of sGC results in decreased responsiveness to NO characterized by loss of NO-stimulated sGC activity. Desensitization of sGC is concentration- and time-dependent on exposure to CSNO, and sensitivity of sGC to NO can be restored and its S-nitrosylation prevented with cellular increase of thiols. We confirm in vitro with semipurified sGC that S-nitrosylation directly causes desensitization, suggesting that other cellular factors are not required. Two potential S-nitrosylated cysteines in the alpha- and beta-subunits of sGC were identified by MS. Replacement of these cysteines, C243 in alpha and C122 in beta, created mutants that were mostly resistant to desensitization. Structural analysis of the region near beta-C122 in the homologous Nostoc H-NOX crystal structure indicates that this residue is in the vicinity of the heme and its S-nitrosylation could dampen NO activation by affecting the positions of key residues interacting with the heme. This study suggests that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells and could be a mechanism of NO tolerance.

View details for DOI 10.1073/pnas.0703944104

View details for Web of Science ID 000248472100016

View details for PubMedID 17636120
NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism EMBO JOURNAL Ma, X., Sayed, N., Beuve, A., van den Akker, F. 2007; 26 (2): 578-588
Abstract

Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.

View details for DOI 10.1038/sj.embol.7601521

View details for Web of Science ID 000243730700028

View details for PubMedID 17215864
Protein kinase G phosphorylates soluble guanylyl cyclase and inhibits its activity Papapetropoulos, A. 2007: P45
View details for DOI 10.1186/14712210
S-nitrosylation of soluble guanylyl cyclase: a novel mechanism of nitrate tolerance? Sayed, N. 2007: 1?1
NO-CGMP Pathway Modulates Actin Remodeling during Neuronal Differentiation AMER SOC CELL BIOLOGY Sayed, N. 2006

Nazish Sayed MD, PhD

Assistant Professor (Research) of Surgery (Vascular Surgery) and at the Stanford Cardiovascular Institute

Surgery - Vascular Surgery

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