Winston Shim

Angiopoietin: A TIE(d) Balance in Tumor Angiogenesis

Angiopoietins (ANG-1 and ANG-2) and their TIE-2 receptor tyrosine kinase have wide-ranging effects on tumor malignancy that includes angiogenesis, inflammation, and vascular extravasation. These multifaceted pathways present a valuable opportunity in developing novel inhibition strategies for cancer treatment. However, the regulatory role of ANG-1 and ANG-2 in tumor angiogenesis remains controversial. There is a complex interplay between complementary yet conflicting roles of both the ANGs in shaping the outcome of angiogenesis. Embryonic vascular development suggests that ANG-1 is crucial in engaging interaction between endothelial and perivascular cells. However, recruitment of perivascular cells by ANG-1 has recently been implicated in its antiangiogenic effect on tumor growth. It is becoming clear that TIE-2 signaling may function in a paracrine and autocrine manner directly on tumor cells because the receptor has been increasingly found in tumor cells. In addition, α5β1 and αvβ5 integrins were recently recognized as functional receptors for ANG-1 and ANG-2. Therefore, both the ligands may have wide-ranging functions in cellular activities that affect overall tumor development. Collectively, these TIE-2–dependent and TIE-2–independent activities may account for the conflicting findings of ANG-1 and ANG-2 in tumor angiogenesis. These uncertainties have impeded development of a clear strategy to target this important angiogenic pathway. A better understanding of the molecular basis of ANG-1 and ANG-2 activity in the pathophysiologic regulation of angiogenesis may set the stage for novel therapy targeting this pathway. (Mol Cancer Res 2007;5(7):655–65)

Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder associated with sudden cardiac death. Its pathophysiology is still poorly understood. We aimed to produce an in vitro cellular model of ARVC using patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes and determine whether the model could recapitulate key features of the disease phenotype. METHODS AND RESULTS Dermal fibroblasts were obtained from a 30-year-old man with a clinical diagnosis of ARVC, harbouring a plakophilin 2 (PKP2) gene mutation. Four stable iPSC lines were generated using retroviral reprogramming, and functional cardiomyocytes were derived. Gene expression levels of desmosomal proteins (PKP2 and plakoglobin) in cardiomyocytes from ARVC-iPSCs were significantly lower compared with cardiomyocytes from control iPSCs (P< 0.01); there were no significant differences in the expression of desmoplakin, N-cadherin, and connexin 43 between the two groups. Cardiomyocytes derived from ARVC-iPSCs exhibited markedly reduced immunofluorescence signals when stained for PKP2 and plakoglobin, but similar levels of staining for desmoplakin, N-cadherin, and connexin 43 compared with control cardiomyocytes. Transmission electron microscopy showed that ARVC-iPSC cardiomyocytes were larger and contained darker lipid droplets compared with control cardiomyocytes. After 2 weeks of cell exposure to adiopgenic differentiation medium, ARVC-iPSC cardiomyocytes were found to contain a significantly greater amount of lipid, calculated using Oil Red O staining, compared with controls (734 ± 35.6 vs. 8.1 ± 0.49 a.u., respectively; n = 7, P = 0.001). CONCLUSION Patient-specific iPSC-derived cardiomyocytes display key features of ARVC, including reduced cell surface localization of desmosomal proteins and a more adipogenic phenotype.

Modeling type 3 long QT syndrome with cardiomyocytes derived from patient-specific induced pluripotent stem cells

BACKGROUND Type 3 long QT syndrome (LQT3) is the third most common form of LQT syndrome and is characterized by QT-interval prolongation resulting from a gain-of-function mutation in SCN5A. We aimed to establish a patient-specific human induced pluripotent stem cell (hiPSC) model of LQT3, which could be used for future drug testing and development of novel treatments for this inherited disorder. METHODS AND RESULTS Dermal fibroblasts obtained from a patient with LQT3 harboring a SCN5A mutation (c.5287G>A; p.V1763M) were reprogrammed to hiPSCs via repeated transfection of mRNA encoding OCT-4, SOX-2, KLF-4, C-MYC and LIN-28. hiPSC-derived cardiomyocytes (hiPSC-CMs) were obtained via cardiac differentiation. hiPSC-CMs derived from the patients healthy sister were used as a control. Compared to the control, patient hiPSC-CMs exhibited dominant mutant SCN5A allele gene expression, significantly prolonged action potential duration or APD (paced CMs of control vs. patient: 226.50 ± 17.89 ms vs. 536.59 ± 37.1 ms; mean ± SEM, p < 0.005), an increased tetrodotoxin (TTX)-sensitive late or persistent Na(+) current (control vs. patient: 0.65 ± 0.11 vs. 3.16 ± 0.27 pA/pF; n = 9, p < 0.01), a positive shift of steady state inactivation and a faster recovery from inactivation. Mexiletine, a NaV1.5 blocker, reversed the elevated late Na(+) current and prolonged APD in LQT3 hiPSC-CMs. CONCLUSIONS We demonstrate that hiPSC-CMs derived from a LQT3 patient recapitulate the biophysical abnormalities that define LQT3. The clinical significance of such an in vitro model is in the development of novel therapeutic strategies and a more personalized approach in testing drugs on patients with LQT3.

Pharmacological response of human cardiomyocytes derived from virus-free induced pluripotent stem cells

AIMS Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes. METHODS AND RESULTS The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose-response (0.1-10 µM) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 µM) suppressed the response to isoprenaline, while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 µM) reduced the corrected field potential duration by 45%, tetrodotoxin (10 µM) shortened the minimal field potential by 40%, and E-4031 (50 nM) prolonged field repolarization. CONCLUSION Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine.

Re-trafficking of herg reverses long QT syndrome 2 phenotype in human iPS-derived cardiomyocytes

AIMS Long QT syndrome 2 (LQTS2) caused by missense mutations in hERG channel is clinically associated with abnormally prolonged ventricular repolarization and sudden cardiac deaths. Modelling monogenic arrhythmogenic diseases using human-induced pluripotent stem cells (hiPSCs) offers unprecedented mechanistic insights into disease pathogenesis. We utilized LQTS2-hiPSC-derived cardiomyocytes (CMs) to elucidate pathological changes and to demonstrate reversal of LQTS2 phenotype in a therapeutic intervention using a pharmacological agent, (N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine) (ALLN). METHODS AND RESULTS We generated LQTS2-specific CMs (A561V missense mutation in KCNH2) from iPSCs using the virus-free reprogramming method. These CMs recapitulate dysfunction of hERG potassium channel with diminished IKr currents, prolonged repolarization durations, and elevated arrhythmogenesis due to reduced membrane localization of glycosylated/mature hERG. Dysregulated expression of folding chaperones and processing proteasomes coupled with sequestered hERG in the endoplasmic reticulum confirmed trafficking-induced disease manifestation. Treatment with ALLN, not only increased membrane localization of mature hERG but also reduced repolarization, increased IKr currents and reduced arrhythmogenic events. Diverged from biophysical interference of hERG channel, our results show that modulation of chaperone proteins could be therapeutic in LQTS2 treatment. CONCLUSION Our in vitro study shows an alternative approach to rescue diseased LQTS2 phenotype via corrective re-trafficking therapy using a small chemical molecule, such as ALLN. This potentially novel approach may have ramifications in other clinically relevant trafficking disorders.

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair.

Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice.

Angiopoietin‐1 (Ang1) is an angiogenic growth factor that functions through activation of its endothelium‐specific tyrosine kinase receptor Tie2; it mediates the interaction between endothelial and surrounding cells to promote the remodeling, maturation and stabilization of blood vessels. Although Ang1 is expressed constitutively in many adult tissues, its role in tumor growth and metastasis is not clear. Here we describe experiments in which Ang1 expression was inhibited in HeLa cells by an antisense RNA approach. The modified HeLa cells produced significantly less Ang1 protein both in cultured cells and in tumors formed when these cells were injected into immunodeficient mice. The Ang1 antisense tumors grew much more slowly, with significantly reduced tumor angiogenesis compared with control tumors. Furthermore, they also had substantially increased tumor cell apoptosis and decreased tumor necrosis. Our results indicate that the perturbation of Ang1 expression in tumors could be an effective method to control tumor growth by inhibiting tumor angiogenesis and that antisense RNA is an efficient way to inhibit Ang1 protein production in tumor cells.

Electrophysiology of human cardiac atrial and ventricular telocytes

Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber‐specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole‐cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c‐Kit and PDGFR‐β were identified. These cells expressed large conductance Ca2+‐activated K+ current (BKCa) and inwardly rectifying K+ current (IKir), but not transient outward K+ current (Ito) and ATP‐sensitive potassium current (KATP). The active channels were functionally competent with demonstrated modulatory response to H2S and transforming growth factor (TGF)‐β1 whereby H2S significantly inhibited the stimulatory effect of TGF‐β1 on current density of both BKCa and IKir. Furthermore, H2S attenuated TGF‐β1‐stimulated KCa1.1/Kv1.1 (encode BKCa) and Kir2.1 (encode IKir) expression in TCs. Our results show that functionally competent K+ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.

G-CSF for stem cell therapy in acute myocardial infarction: friend or foe?

Stem cell-based therapy has emerged as a potential therapeutic option for patients with acute myocardial infarction. The ability of granulocyte colony-stimulating factor (G-CSF) to mobilize endogenous stem cells as well as to protect cardiomyocytes at risk via paracrine effects has attracted considerable attention. In the past decade, a number of clinical trials were carried out to study the efficacy of G-CSF in cardiac repair. These trials showed variable outcomes in terms of improved cardiac contractile function and suppressed left ventricular negative remodelling. Critical examinations of these results have raised doubts concerning the effectiveness of G-CSF in modulating functional recovery. However, these cumulative clinical experiences are helpful in the understanding of mechanisms and roles of signalling pathways in regulating homing and engraftment of bone marrow stem cells to the infarcted heart. In this review, we discuss some of the observations that may have influenced the clinical outcomes. Improving strategies that target the critical aspects of G-CSF-driven cardiac therapy may provide a better platform to augment clinical benefits in future trials.

Pharmacoelectrophysiology of Viral-Free Induced Pluripotent Stem Cell–Derived Human Cardiomyocytes

Development of pharmaceutical agents for cardiac indication demands elaborate safety screening in which assessing repolarization of cardiac cells remains a critical path in risk evaluations. An efficient platform for evaluating cardiac repolarization in vitro significantly facilitates drug developmental programs. In a proof of principle study, we examined the effect of antiarrhythmogenic drugs (Vaughan Williams class I-IV) and noncardiac active drugs (terfenadine and cisapride) on the repolarization profile of viral-free human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Extracellular field potential (FP) recording using microelectrode arrays demonstrated significant delayed repolarization as prolonged corrected FP durations (cFPDs) by class I (quinidine and flecainide), class III (sotalol and amiodarone), and class IV (verapamil), whereas class II drugs (propranolol and nadolol) had no effects. Consistent with their sodium channel-blocking ability, class I drugs also significantly reduced FPmin and conduction velocity. Although lidocaine (class IB) had no effects on cFPDs, verapamil shortened cFPD and FPmin by 25 and 50%, respectively. Furthermore, verapamil reduced beating frequencies drastically. Importantly, the examined drugs exhibited dose-response curve on prolongation of cFPDs at an effective range that correlated significantly with therapeutic plasma concentrations achieved clinically. Consistent with clinical outcomes, drug-induced arrhythmia of tachycardia and bigeminy-like waveforms by quinidine, flecainide, and sotalol was demonstrated at supraphysiological concentrations. Furthermore, off-target effects of terfenadine and cisapride on cFPD and Na( + ) channel blockage were similarly revealed. These results suggest that hiPSC-CMs may be useful for safety evaluation of cardioactive and noncardiac acting drugs for personalized medicine.