Kihwan Kwon

Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow.

Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

Quantitative analysis of peripheral tissue perfusion using spatiotemporal molecular dynamics.

Background Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging. Methodology/Principal Findings We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1. Conclusions/Significance We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis.

Unsorted human adipose tissue-derived stem cells promote angiogenesis and myogenesis in murine ischemic hindlimb model.

We examined the protective effect of unsorted human adipose tissue-derived stem cells (hADSCs) with a short-term culture in endothelial differentiation medium on tissue repair after ischemic injury. hADSCs were isolated from human subcutaneous adipose tissue and cultured in vitro in endothelial differentiation medium for 2wks before transplantation. Cultured hADSCs showed a typical mesenchymal stromal cell-like phenotype, positive for endothelial-specific markers including VE-cadherin, Flt-1, eNOS, and vWF but not CD31. Two hours after ligation of the femoral artery and vein, mice were injected with the unselected hADSCs locally near the surgery site and tested for tissue perfusion and repair. Tissue perfusion rates of the ischemic limbs were significantly higher in the group treated with hADSCs compared with those of the control mice as early as post-operative day 3 (median 195.3%/min; interquartile range, 82.0-321.1 vs. median 47.1%/min; interquartile range, 18.0-58.7; p=0.001 by Friedman two-way analysis). Subsequently, the mice treated with hADSC showed better prognosis at 4wks after surgery, and the histological analysis revealed increased vascular density and reduced muscle atrophy in the hADSC-transplanted limbs. Moreover, hADSC-treated muscle contained differentiated myocytes positive for human NF-κB and myogenin antigen. These results collectively indicate that unsorted hADSCs after a 2-wk-in vitro culture have a therapeutic potential in ischemic tissue injury via inducing both angiogenesis and myogenesis.

GTP Cyclohydrolase I Phosphorylation and Interaction With GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide

Rationale: GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH4 production in response to laminar shear. Objective: We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. Methods and Results: Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo. Conclusions: These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH4 to be produced in response to laminar shear stress.

Dynamic fluorescence imaging of indocyanine green for reliable and sensitive diagnosis of peripheral vascular insufficiency

Quantitative measurement of functional tissue perfusion is essential for early diagnosis and proper treatment of peripheral arterial occlusive disease (PAOD). We have previously demonstrated that dynamic imaging of near-infrared fluorophore indocyanine green (ICG) can be a noninvasive and sensitive tool to measure tissue perfusion. In the present study, we investigated the clinical efficacy of ICG perfusion imaging method for the diagnosis of PAOD. Total nineteen PAOD patients and age-matched controls (n=10) were evaluated for lower extremity tissue perfusion using ICG perfusion imaging. The perfusion rates of the lower extremities with severe PAOD (n=25 legs, 16.6±8.3%/min) were significantly lower than those of normal controls (38.1±17.3%/min, p<0.001). In cases of mild PAOD, the perfusion rates (n=11 legs, 18.3±10.3%/min) were also significantly lower compared to the control; while the conventional ankle-brachial index (ABI) test failed to detect mild functional impairment. These results collectively indicate that ICG perfusion imaging can be a very effective tool for diagnosis of PAOD with a superior efficacy in comparison to conventional ABI test.

Extracorporeal shock wave stimulates expression of the angiogenic genes via mechanosensory complex in endothelial cells: mimetic effect of fluid shear stress in endothelial cells.

BACKGROUND Extracorporeal shock wave has been used in the noninvasive treatment of various diseases including musculoskeletal disorders. In particular, shock wave with low energy level showed anti-inflammatory effect and increased angiogenesis in ischemic tissues. However, the detailed cellular pathway in endothelial signaling is not fully understood. We investigate the role of shock wave with low energy level in angiogenic gene expression and underlying molecular mechanism by comparing the laminar and oscillatory fluid shear stresses in endothelial cells. METHODS AND RESULTS We show that shock wave with low energy level (0.012-0.045 mJ/mm(2)) stimulated phosphorylation of Akt, eNOS and Erk 1/2 in a time-dependent manner which is similar to the effect of laminar fluid shear stress. The transfection of endothelial cells with siRNA encoding VEGFR2, VE-cadherin and PECAM-1 inhibited shock wave-induced phosphorylation of Akt, eNOS and Erk 1/2 and angiogenic gene expressions, including Akt, eNOS, KLF2/4, and Nur77. Moreover, mechanical stimulation through extracorporeal shock wave induced endothelial cell migration and tube formation. CONCLUSIONS Our results demonstrate that shock wave-induced Akt/eNOS phosphorylation and angiogenic gene expression were mediated through the mechanosensory complex formation involving VEGFR-2, VE-cadherin and PECAM-1 which was similar to the effect of laminar shear stress.

Application of novel dynamic optical imaging for evaluation of peripheral tissue perfusion

Measurement of functional tissue perfusion should be needed for preventive measures, early diagnosis, and adequate treatment especially in the patients with peripheral vascular diseases (PVD). Significant attention has also been given to in vivo near-infrared (NIR) fluorescence imaging because of deep tissue penetration due to low absorbance and scattering. In this study, a new method, indocyanine green (ICG) perfusion imaging to evaluate the peripheral tissue perfusion that employs the intravenous injection of ICG dye, planar imaging with a CCD digital imaging system, and analysis of spatiotemporal dynamics have been applied to diagnose perfusion rate of human lower extremities from a normal and a PVD case. The feet tissue perfusions of the PVD patient were measured as 25% to 50% compared with those of normal feet tissue. The diagnostic result indicates that ICG perfusion imaging method is sensitive enough to diagnose PVD and take noninvasive monitoring treatment effects of peripheral vascular diseases in clinical setting.

Vascular Injury Involves the Overoxidation of Peroxiredoxin Type II and Is Recovered by the Peroxiredoxin Activity Mimetic That Induces Reendothelialization

Background— Typical 2-Cys peroxiredoxin (Prx) is inactivated by overoxidation of the peroxidatic cysteine residue under oxidative stress. However, the significance in the context of vascular disease is unknown. Methods and Results— Immunohistochemical analyses revealed that 2-Cys Prxs, particularly Prx type II, are heavily overoxidized in balloon-injured rodent carotid vessels and in human atherosclerotic lesions. Consistent with this observation, the selective depletion of Prx II exacerbated neointimal hyperplasia in injured carotid vessels. We also found that the epipolythiodioxopiperazine class of fungal metabolites exhibited an enzyme-like activity mimicking 2-Cys Prx peroxidase and manifestly eliminated the intracellular H2O2 in the vascular cells. Functionally, the epipolythiodioxopiperazines reciprocally regulated the platelet-derived growth factor receptor-&bgr;– and vascular endothelial growth factor receptor–mediated signaling in these vascular cells by replacing Prx II. As a consequence, the epipolythiodioxopiperazines inhibited the proliferative and migratory activities of smooth muscle cells but promoted those of endothelial cells in vitro. Moreover, administration of the epipolythiodioxopiperazines to the injured carotid vessels resulted in a successful recovery by inhibiting neointimal hyperplasia without causing cytotoxicity and simultaneously inducing reendothelialization. Conclusions— This study reveals for the first time the involvement of the 2-Cys Prx overoxidation and thus the therapeutic use of their activity mimetic in vascular injuries like stenting.

Clinical implication of an impaired fasting glucose and prehypertension related to new onset atrial fibrillation in a healthy Asian population without underlying disease: a nationwide cohort study in Korea

Aims For healthy populations without comorbidities, whether prehypertension and impaired fasting glucose (IFG) are associated with new onset atrial fibrillation (AF) is not well known. Methods and results We included 366 507 subjects (age ≥20 years) not diagnosed with non-valvular AF from the Korean National Health Insurance Service-National Sample Cohort (NHIS-NSC) from 2003 to 2008. In total, 139 306 subjects diagnosed with AF-related comorbidities were excluded, and a 227 102 healthy population was followed up until 2013. The body mass index (BMI), blood pressure (BP), and fasting blood glucose (BG) level were acquired during National health check-ups. Subjects with IFG [hazard ratio (HR) 1.16, P = 0.017] had a higher AF risk and the diastolic BP (HR 1.11, P = 0.045) was a stronger indicator for an AF incidence than the systolic BP. After dividing the subjects into two mutually exclusive groups, AF incidence was increased dramatically by the combination effect of both prehypertension and an IFG in BMI <25 kg/m2 group, but, in BMI ≧25 kg/m2 group, did not show this tendency. An IFG related to AF risk was more prominent in the BMI <25 kg/m2 population (HR 1.18, P = 0.025) than those with a BMI ≥25 kg/m2, and subjects with both an IFG and prehypertension had a greater AF risk (HR 1.27, P = 0.016) than those without. Conclusion Even in a healthy Asian populations without comorbidities, prehypertension and IFG were important risk factors of AF. Specifically, when prehypertension, including systolic and diastolic BPs, was finally combined with the IFG, the risk of new onset AF was increased especially in the BMI <25 kg/m2 group.

Accelerated Thrombotic Occlusion of a Medium-Sized Coronary Aneurysm in Kawasaki Disease by the Inhibitory Effect of Ibuprofen on Aspirin

The fate of coronary artery aneurysms (CAAs) caused by Kawasaki disease depends mainly on their initial size and shape. Small to medium-sized CAAs are known to regress to normal size or decrease in size, with a good outcome. A patient with Kawasaki disease is reported who had a medium-sized CAA prematurely occluded with thrombi during regression, resulting in myocardial ischemia. This event was probably due to simultaneous use of aspirin and ibuprofen. Thus, the concomitant use of ibuprofen should be avoided when aspirin is given as an antiplatelet agent because ibuprofen blocks the platelet inhibition induced by aspirin.