Chun-Hsien Wu

Mesenchymal Stem Cells from Human Umbilical Cord Express Preferentially Secreted Factors Related to Neuroprotection, Neurogenesis, and Angiogenesis

Mesenchymal stem cells (MSCs) are promising tools for the treatment of diseases such as infarcted myocardia and strokes because of their ability to promote endogenous angiogenesis and neurogenesis via a variety of secreted factors. MSCs found in the Wharton’s jelly of the human umbilical cord are easily obtained and are capable of transplantation without rejection. We isolated MSCs from Wharton’s jelly and bone marrow (WJ-MSCs and BM-MSCs, respectively) and compared their secretomes. It was found that WJ-MSCs expressed more genes, especially secreted factors, involved in angiogenesis and neurogenesis. Functional validation showed that WJ-MSCs induced better neural differentiation and neural cell migration via a paracrine mechanism. Moreover, WJ-MSCs afforded better neuroprotection efficacy because they preferentially enhanced neuronal growth and reduced cell apoptotic death of primary cortical cells in an oxygen-glucose deprivation (OGD) culture model that mimics the acute ischemic stroke situation in humans. In terms of angiogenesis, WJ-MSCs induced better microvasculature formation and cell migration on co-cultured endothelial cells. Our results suggest that WJ-MSC, because of a unique secretome, is a better MSC source to promote in vivo neurorestoration and endothelium repair. This study provides a basis for the development of cell-based therapy and carrying out of follow-up mechanistic studies related to MSC biology.

Dysregulated miR-361-5p/VEGF Axis in the Plasma and Endothelial Progenitor Cells of Patients with Coronary Artery Disease

Dysfunction and reduction of circulating endothelial progenitor cell (EPC) is correlated with the onset of cardiovascular disorders including coronary artery disease (CAD). VEGF is a known mitogen for EPC to migrate out of bone marrow to possess angiogenic activities, and the plasma levels of VEGF are inversely correlated to the progression of CAD. Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. However, how miRNAs and VEGF cooperate to regulate CAD progression is still unclear. Through the small RNA sequencing (smRNA-seq), we deciphered the miRNome patterns of EPCs with different angiogenic activities, hypothesizing that miRNAs targeting VEGF must be more abundant in EPCs with lower angiogenic activities. Candidates of anti-VEGF miRNAs, including miR-361-5p and miR-484, were enriched in not only diseased EPCs but also the plasma of CAD patients. However, we found out only miR-361-5p, but not miR-484, was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-361-5p to the 3′-UTR of VEGF mRNA. Knock down of miR-361-5p not only restored VEGF levels and angiogenic activities of diseased EPCs in vitro, but further promoted blood flow recovery in ischemic limbs of mice. Collectively, we discovered a miR-361-5p/VEGF-dependent regulation that could help to develop new therapeutic modalities not only for ischemia-related diseases but also for tumor angiogenesis.

miRNome traits analysis on endothelial lineage cells discloses biomarker potential circulating microRNAs which affect progenitor activities

BackgroundEndothelial progenitor cells (EPCs) play a fundamental role in not only blood vessel development but also post-natal vascular repair. Currently EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Both EPC types assist angiogenesis and have been linked to ischemia-related disorders, including coronary artery disease (CAD).ResultsWe found late EPCs are more mobile than early EPCs and matured endothelial cells (ECs). To pinpoint the mechanism, microRNA profiles of early EPCs late EPCs, and ECs were deciphered by small RNA sequencing. Obtained signatures made up of both novel and known microRNAs, in which anti-angiogenic microRNAs such as miR-221 and miR-222 are more abundant in matured ECs than in late EPCs. Overexpression of miR-221 and miR-222 resulted in the reduction of genes involved in hypoxia response, metabolism, TGF-beta signalling, and cell motion. Not only hamper late EPC activities in vitro, both microRNAs (especially miR-222) also hindered in vivo vasculogenesis in a zebrafish model. Reporter assays showed that miR-222, but not miR-221, targets the angiogenic factor ETS1. In contrast, PIK3R1 is the target of miR-221, but not miR-222 in late EPCs. Clinically, both miR-221-PIK3R1 and miR-222-ETS1 pairs are deregulated in late EPCs of CAD patients.ConclusionsOur results illustrate EPCs and ECs exploit unique miRNA modalities to regulate angiogenic features, and explain why late EPC levels and activities are reduced in CAD patients. These data will further help to develop new plasma biomarkers and therapeutic approaches for ischemia-related diseases or tumor angiogenesis.

Differential Expression of Distinct Surface Markers in Early Endothelial Progenitor Cells and Monocyte-Derived Macrophages

Bone marrow-derived endothelial progenitor cells (EPCs) play a fundamental role in postnatal angiogenesis. Currently, EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Reports have shown that early EPCs share common properties and surface markers with adherent blood cells, especially CD14+ monocytes. Distinguishing early EPCs from circulating monocytes or monocyte-derived macrophages (MDMs) is therefore crucial to obtaining pure endothelial populations before they can be applied as part of clinical therapies. We compared the gene expression profiles of early EPCs, blood cells (including peripheral blood mononuclear cells, monocytes, and MDMs), and various endothelial lineage cells (including mature endothelial cells, late EPCs, and CD133+ stem cells). We found that early EPCs expressed an mRNA profile that showed the greatest similarity to MDMs than any other cell type tested. The functional significance of this molecular profiling data was explored by Gene Ontology database search. Novel plasma membrane genes that might potentially be novel isolation biomarkers were also pinpointed. Specifically, expression of CLEC5A was high in MDMs, whereas early EPCs expressed abundant SIGLEC8 and KCNE1. These detailed mRNA expression profiles and the identified functional modules will help to develop novel cell isolation approaches that will allow EPCs to be purified; these can then be used to target cardiovascular disease, tumor angiogenesis, and various ischemia-related diseases.

Dysregulation of Vascular Endothelial Growth Factor Receptor-2 by Multiple miRNAs in Endothelial Colony-Forming Cells of Coronary Artery Disease

Background/Aims: Endothelial colony-forming cells (ECFCs) have the potential to be used in regenerative medicine. Dysfunction of ECFCs is correlated with the onset of cardiovascular disorders, especially coronary artery disease (CAD). Binding of vascular endothelial growth factor A (VEGFA) to vascular endothelial growth factor receptor-2 (VEGFR2) triggers cell motility and angiogenesis of ECFCs, which are crucial to vascular repair. Methods: To identify the miRNA-VEGFR2-dependent regulation of ECFC functions, ECFCs isolated from peripheral blood of disease-free and CAD individuals were subjected to small RNA sequencing for identification of anti-VEGFR2 miRNAs. The angiogenic activities of the miRNAs were determined in both in vitro and in vivo mice models. Results: Three miRNAs, namely miR-410-3p, miR-497-5p, and miR-2355-5p, were identified to be upregulated in CAD-ECFCs, and VEGFR2 was their common target gene. Knockdown of these miRNAs not only restored the expression of VEGFR2 and increased angiogenic activities of CAD-ECFCs in vitro, but also promoted blood flow recovery in ischemic limbs in vivo. miR-410-3p, miR-497-5p, and miR-2355-5p could serve as potential biomarkers for CAD detection as they are highly expressed in the plasma of CAD patients. Conclusions: This modulation could help develop new therapeutic modalities for cardiovascular diseases and other vascular dysregulated diseases, especially tumor angiogenesis.

Modulation of both activator protein-1 and nuclear factor-kappa B signal transduction of human T cells by amiodarone

Amiodarone, a common and effective antiarrhythmic drug, has been reported to have anti-inflammatory effects such as reducing the activation and movement of neutrophils. However, its effects on human T cells remain unclear. The aim of this study was to elucidate the effects and possible underlying mechanisms of amiodarone on human T cells. We isolated human primary T cells from the peripheral blood of healthy volunteers and performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, electrophoretic mobility shift assay, luciferase assay, and Western blotting to evaluate the modulatory effects of amiodarone on human T cells. We found that amiodarone dose dependently inhibited the production of cytokines, including interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha, and interferon-gamma in activated human T cells. By flow cytometry, we demonstrated that amiodarone suppressed the expression of IL-2 receptor-alpha (CD25) and CD69, the cell surface markers of activated T cells. Moreover, molecular investigations revealed that amiodarone down-regulated activator protein-1 (AP-1) and nuclear factor kappa-B (NF-κB) DNA-binding activities in activated human T cells and also inhibited DNA binding and transcriptional activities of both AP-1 and NF-κB in Jurkat cells. Finally, by Western blotting, we showed that amiodarone reduced the activation of c-Jun NH2-terminal protein kinase and P38 mitogen-activated protein kinase, and suppressed stimuli-induced I-kappa B-alpha degradation in activated human T cells. Through regulation of AP-1 and NF-κB signaling, amiodarone inhibits cytokine production and T cell activation. These results show the pleiotropic effects of amiodarone on human T cells and suggest its therapeutic potential in inflammation-related cardiovascular disorders.

Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients

Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases.

Stimulatory Influences of Far Infrared Therapy on the Transcriptome and Genetic Networks of Endothelial Progenitor Cells Receiving High Glucose Treatment.

BACKGROUND Endothelial progenitor cells (EPCs) play a fundamental role in vascular repair and angiogenesis- related diseases. It is well-known that the process of angiogenesis is faulty in patients with diabetes. Long-term exposure of peripheral blood EPCs to high glucose (HG-EPCs) has been shown to impair cell proliferation and other functional competencies. Far infrared (FIR) therapy can promote ischemia-induced angiogenesis in diabetic mice and restore high glucose-suppressed endothelial progenitor cell functions both in vitro and in vivo. However, the detail mechanisms and global transcriptome alternations are still unclear. METHODS In this study, we investigated the influences of FIR upon HG-EPC gene expressions. EPCs were obtained from the peripheral blood and treated with high glucose. These cells were then subjected to FIR irradiation and functional assays. RESULTS Those genes responsible for fibroblast growth factors, Mitogen-activated protein kinases (MAPK), Janus kinase/signal transducer and activator of transcription and prostaglandin signaling pathways were significantly induced in HG-EPCs after FIR treatment. On the other hand, mouse double minute 2 homolog, genes involved in glycogen metabolic process, and genes involved in cardiac fibrosis were down-regulated. We also observed complex genetic networks functioning in FIR-treated HG-EPCs, in which several genes, such as GATA binding protein 3, hairy and enhancer of split-1, Sprouty Homolog 2, MAPK and Sirtuin 1, acted as hubs to maintain the stability and connectivity of the whole genetic network. CONCLUSIONS Deciphering FIR-affected genes will not only provide us with new knowledge regarding angiogenesis, but also help to develop new biomarkers for evaluating the effects of FIR therapy. Our findings may also be adapted to develop new methods to increase EPC activities for treating diabetes-related ischemia and metabolic syndrome-associated cardiovascular disorders. KEY WORDS Endothelial progenitor cell; Far infrared; Microarray; Systems biology.

Clinical and prognostic correlates of ST-elevation myocardial infarction patients with normal coronary angiography

Background: Revascularization within a 90-min door-to-balloon time is a strict policy enacted in Taiwan. Prompt diagnosis is critical to avoid an unnecessary procedure and catheterization laboratory activation. This study was aimed to investigate the clinical and prognostic characteristics of the patients with ST-elevation myocardial infarction (STEMI) referred for primary percutaneous coronary intervention (PCI) and normal coronary arteries found following coronary angiography (CAG). Materials and Methods: From October 2009 to December 2012, 216 consecutive patients with STEMI referred for primary PCI were enrolled. The data of clinical history, physical examination, laboratory results, electrocardiography, echocardiography, CAG findings, diagnosis, and outcomes were collected and analyzed. Results: A total of 17 patients were proved normal coronaries angiographically. The incidence of the conditions mimicking as STEMI is 7.9%. Alternative diagnosis was coronary spasm (n = 7), peri-myocarditis (n = 6), apical ballooning syndrome (n = 3), anaphylactic shock (n = 1). Compared with STEMI group, patients in normal coronaries group were younger, with a less premature family history of coronary artery disease (CAD), and reported angina. The 30-day mortality rate in the normal coronaries group was 5.9%. Conclusions: Cautiously evaluating CAD risk factors and symptoms of angina and awareness of alternative diagnosis are important to make a prompt diagnosis without compromising accuracy in the patients presenting as suspected STEMI.

Critical hyperkalaemia mimicking acute cerebral infarction in a haemodialysis patient

An ambulatory 66-year-old uraemic woman on chronic haemodialysis presented to the emergency department with progressive left-sided weakness for 10 h. Other histories were non-contributory. On admission, she was alert and fully oriented with normal language function. Body temperature was 36.6°C, heart rate 56 b.p.m., respiratory rate 18 breaths/min and blood pressure 177/50 mmHg. Neurological examinations revealed left hemiparesis and hyporeflexia with muscle strength grades of 2/5 and 4/5, with relatively normal tendon reflexes in the right limbs. Cranial and sensory nerves were intact. Emergency brain computed tomography revealed an old lacunar infarction in the right basal ganglion with no other active organic lesions. Laboratory tests revealed marked hyperkalaemia with a serum potassium level of 9.7 mmol/L and electrocardiography also showed typical findings of hyperkalaemia. Other serum biochemistries were as follows: sodium, 134 mmol/L; chloride, 102 mmol/L; glucose, 7.0 mmol/L; urea nitrogen, 32.5 mmol/L; creatinine, 645.3 μmol/L; calcium, 2.4 mmol/L and creatinine phosphokinase, 137 IU/L. Immediate potassium lowering therapy combined with haemodialysis using low potassium dialysate (1.0 mmol/L) rapidly corrected electrocardiographic abnormalities, hyperkalaemia and muscle strength within 1 h (Fig. 1). She was taking long-term prescription medications, including isosorbide mononitrate, calcium acetate, furosemide and sennoside. On reviewing her food history, it was determined that she ate three sugar apples and half a durian (both high potassium fruits) in the past 2 days, which contributed to her severe hyperkalaemia. She was discharged from the hospital after haemodialysis. There were no neuromuscular sequelae at 6-month follow up at the outpatient department. Potentially life-threatening cardiac dysrhythmias are well-known complications of severe hyperkalaemia, worsening the associated morbidity and mortality. However, neuromuscular presentations, such as quadriparesis, weakness or myalgias may sometimes emerge as the predominant symptom before cardiac complaints. In this patient, acute unilateral paralysis and an infarct-like lesion in the right basal ganglion may alert clinicians to the early diagnosis of acute cerebral infarction. However, neither ongoing neurological symptoms nor any neuromuscular sequelae for months made cerebral infarction, transient ischaemic attack or myelopathy unlikely. In contrast, strong correlations between hemiparesis and severe hyperkalaemia suggested the high probability that paralysis was caused by hyperkalaemia. On rare occasions, metabolic disorders, such as hypoglycaemia and hypokalaemia, may present with unilateral paralysis. Instead of left hemiparesis, slightly decreased muscle strength grading of the right side suggested that paralysis was not strictly unilateral. Acute deterioration of a previous deficit from systemic illness could be a reasonable explanation for the pronounced involvement of the left side and improvement with dialysis. Therefore, we concluded that her silent cerebral infarction predisposed her to predominantly left-sided paralysis in severe hyperkalaemia. Durians and sugar apples are both high-potassium fruits (11.2 mEq and 6.3 mEq potassium per 100 grams of flesh respectively). Her potassium intake was at least 70–90 mEq, which was large enough to lead to severe hyperkalaemia. Figure 1 The correlation of the serum potassium level and corresponding muscle strength during haemodialysis with low potassium dialysate. ( ), Serum potassium level; ( ), muscle strength grading (left); ( ), muscle strength grading (right). bs_bs_banner