Yung-Tsung Chiu

Increased Extracellular Collagen Matrix in Myocardial Sleeves of Pulmonary Veins: An Additional Mechanism Facilitating Repetitive Rapid Activities in Chronic Pacing‐Induced Sustained Atrial Fibrillation

Introduction: Cell uncoupling due to fibrosis or increased extracellular collagen matrix (ECM) affects the formation of ectopic focal activity. Whether or not the increase of ECM also exists in the pulmonary veins (PVs) with rapid atrial pacing is unknown. We sought to test the hypothesis that in chronic atrial pacing dogs with sustained atrial fibrillation (AF), the amount of ECM was increased in both atria and the PVs.

Bronchiolitis obliterans organizing pneumonia in Swine associated with porcine circovirus type 2 infection.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a chronic respiratory disease. Although the pathogenesis of BOOP is still incompletely understood, BOOP is responsive to steroids and has a good prognosis. In our five pigs with chronic postweaning multisystemic wasting syndrome (PMWS), typical BOOP lesions were revealed. All five porcine lungs showed typical intraluminal plugs, and porcine circovirus type 2 (PCV2) was identified. They also exhibited similar pathologic findings such as proliferation of type II pneumocytes and myofibroblasts (MFBs), extracellular collagen matrix (ECM) deposition, and fragmentation of elastic fibers. MFBs migration correlative molecules, for instance, gelatinase A, B and osteopontin, appeared strongly in the progressing marginal area of polypoid intraluminal plugs of fibrotic lesion. These molecules colocalized with the active MFBs. Both gelatinase activity and intercellular level of active MFBs were significantly increased (P < .05). Porcine chronic bronchopneumonia leads to BOOP and it is associated with PCV2 persistent infection. Swine BOOP demonstrates similar cellular constituents with human BOOP. Perhaps their molecular mechanisms of pathogenesis operate in a similar way. Thus we infer that the swine BOOP can be considered as a potential animal model for human BOOP associated with natural viral infection. Moreover, it is more convenient to obtain samples.

Mycophenolate mofetil alleviates lupus nephritis through urokinase receptor signaling in a mice model

Lupus nephritis (LN) is usually associated with widespread effacement of the podocytes’ foot processes leading to proteinuria. Induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via promoting podocytes’ motility and kidney permeability in the glomerulus. Very little is known about uPAR signaling in LN. Mycophenolate mofetil (MMF), an immunosuppressive agent, efficiently modulates the development of LN in humans and mice, but there are no data concerning the direct uPAR involvement on podocytes in LN. The MMF efficiency and uPAR involvement signaling in NZB×NZW F1 lupus-prone mice were examined by proteinuria, renal function and pathology, immune complex deposits, and uPAR expression of podocytes by immunofluorescence staining and quantitative RT-PCR. After MMF treatment, the proteinuria (p < 0.01), BUN level (p < 0.05) and immunodeposition in glomeruli (p < 0.001) were significantly improved. Most important, the renal uPAR mRNA levels (p < 0.001) and uPAR protein level of podocytes (p < 0.001) were significantly reduced. The beneficial effect of MMF on LN could be attributed, at least in part, to the inhibition of uPAR expression in podocytes. These findings demonstrated uPAR could have potential as a predictive index for response to LN therapeutics.

Effect of oxidized regenerated cellulose on the healing of pharyngeal wound: an experimental animal study.

Background: This study aimed to investigate the relationship between oxidized regenerated cellulose (ORC) and mucosa healing in an experimental animal model. Methods: Fifteen adult Sprague‐Dawley rats were randomly divided into three groups that underwent different wound treatments. In Group 1, no pharyngeal wound was created. In Group 2, the pharyngeal wound was sutured with Prolene only. In Group 3, the pharyngeal wound was sutured with Prolene, and covered with one layer of ORC before closure of the skin wound. The animals were euthanized either 5 or 10 days after operation, and wound conditions were inspected and recorded. Specimens including sections of larynx and pharynx/upper esophagus were taken for microscopic and molecular biological examination. Results: The pharyngotomy/esophagotomy wounds achieved good healing outcomes 10 days after operation. Wounds treated with ORC had significantly diminished inflammatory cell infiltration in microscopic examination when compared with that of those without ORC 5 days after operation. The matrix metalloproteinases (MMP) expression level was higher in wounds of Group 2 and Group 3, when compared with that of group 1. In addition, the MMP expression level was lower in the ORC‐treated wounds when compared with that of those without ORC. There was no significant difference in fibroblast proliferation, collagen deposition, endothelin‐1, alpha‐smooth muscle actin, and transforming growth factor beta 1 expression level between wounds treated with ORC and those without ORC. Conclusion: Reduced inflammatory response and decreased MMP expression level was observed in ORC‐treated wounds. Whether ORC facilitates mucosa healing requires further investigation.

Effects of Shugan-Huayu Powder, A Traditional Chinese Medicine, on Hepatic Fibrosis in Rat Model

Shugan-Huayu powder (SHP) has been administered to outpatients with chronic liver disease without clear anti-fibrosis mechanism. To investigate the anti-fibrotic effects of SHP on liver fibrosis in a rat model and in hepatic stellate cells (HSCs) in vitro, rats were gavaged with CCl4 at 1.0 g/kg body weight twice a week for 8 weeks to induce liver fibrosis and the rats were randomly assigned to one of the three groups: -CCl4 alone, low-dose SHP and high-dose SHP. SHP was given by gavages 5 times a week for 8 weeks. Serum, livers and HSCs were assayed for serology, pathology, western blot, zymography and quantitative RT-PCR. Hepatic function improved as decreased serum aspartate aminotransferase and alanine aminotransferase, and collagen deposition and active HSCs were significantly reduced in CCl4-induced liver by SHP treatment. The expression of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta1 (TGF-beta1) mRNA in fibrotic liver showed significant downregulation after SHP treatment. In vitro, inhibition of alpha-smooth muscle actin (alpha-SMA) expression and MMP-2 secretion of active HSCs were also noticed by SHP treatment. SHP has an antifibrotic effect on CCl4-induced liver fibrosis in rats. Anti-fibrotic mechanisms were probably inhibiting activation of HSCs and decreased expression of MMP-2 and TGF-beta1.

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells.

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca(2+) transients and TGF-β-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.

Nitric oxide and glutamate in the dorsal facial area regulate common carotid blood flow in the cat

Nitric oxide (NO) or glutamate stimulation of dorsal facial area (DFA) increases blood flow in the common carotid artery (CCA), which supplies intra-and extra-cranial tissues. Nitrergic fibers and neurons as well as preganglionic cholinergic neurons are present in the DFA. We hypothesized the presence of nitrergic-glutamatergic fibers and preganglionic nitrergic-cholinergic neurons in the DFA that are involved in the regulation of CCA blood flow. In microdialysis studies, perfusion of the DFA with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) increased the glutamate concentration in the dialysate. This effect was abolished by co-perfusion of methylene blue (a guanylyl cyclase inhibitor). Intra-DFA injection of l-arginine (an NO precursor) or glutamate increased CCA blood flow. The l-arginine-induced flow increase was reduced by prior administration of NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor), d-2-amino-5-phosphonopentanoate (d-AP5, a competitive NMDA receptor antagonist), or glutamate diethylester (GDEE, a competitive AMPA receptor antagonist). The glutamate-induced blood flow increase was reduced by prior administration of l-NAME, 7-NI, or methylene blue. The induced increase in CCA blood flow, however, was not affected by endothelial NO synthase inhibitor. The findings indicate that NO-signal transduction within the DFA might cause glutamate release from presynaptic nitrergic-glutamatergic fibres and that the released glutamate activates NMDA/AMPA receptors on preganglionic nitrergic-cholinergic neurons in the nucleus to activate neuronal NO synthase and guanylyl cyclase in the neurons, leading to an increase in CCA blood flow. These findings may be important for developing therapeutic strategies for the diseases associated with CCA blood flow.

High-dose norepinephrine induces disruption of myocardial extracellular matrix and left ventricular dilatation and dysfunction in a novel feline model.

Background: Intravenous norepinephrine (NE) at a dose of 1‐6 μg/kg/minute can induce increased extracellular matrix (ECM) and hypertrophic cardiomyopathy. This study aimed to investigate the effects of a higher dose of NE on cardiac remodeling. Methods: After intraperitoneal urethane‐chloralose anesthesia, 7 cats (3.03 ± 0.58 kg) received intravenous infusion of NE 30 (ig/kg/minute for 3 hours. Aortic blood pressure and heart rate (HR) were measured by polygraphy at 0, 5,15, 30, 60, 90, 120, and 180 minutes. Left ventricular size and ejection fraction (EF) were measured by M‐mode echocardio‐graphy before and after NE administration. Histopathology was performed by hematoxylin‐eosin, silver impregnation, and Sirius red staining. Activity of matrix metalloproteinases (MMP) in the left ventricle was measured by zymography. Results: Mean blood pressure (mmHg) increased from 139 ± 20 to 198 ± 19,187 ± 23, and 166 ± 16 at 15, 30, and 60 minutes, respectively, during NE infusion. HR (beats/minute) decreased from 214 ± 10 to 158 ± 28 at 15 minutes and then recovered gradually. The left ventricles showed significant dilatation (end‐diastolic diameter: from 1.20 ± 0.18 to 1.58 ± 0.23cm, p = 0.003; end‐systolic diameter: from 0.62 ± 0.23 to 1.35 ± 0.29cm, p = 0.002) and hypokinesia (EF: from 86.2 ± 5.2 to 33.1 ± 16.5%, p< 0.001). Histopathology revealed that left ventricular myocytes were elongated, wavy, and fragmented, while collagen fibers were overstretched, straightened, and disrupted. MMP‐9 activity was significantly elevated (p = 0.003 vs. control), while MMP‐2 activity was unchanged. Conclusion: High‐dose NE increases MMP‐9 activity and causes ECM disruption, left ventricular dilatation and dysfunction.

Effects of mycophenolate mofetil on cutaneous lupus erythematosus in (NZB × NZW) F1 mice.

Background: Few studies have evaluated the effects and precise molecular mechanism of mycophenolate mofetil (MMF) in the treatment of human cutaneous lupus erythematosus (CLE). Our findings shed light on the therapeutic effects of MMF in a UVB‐induced NZB × NZW (NZBW) F1 CLE mouse model. Methods: Continuous MMF treatment (60 mg/kg/day) was administered up to Day 50 from the beginning of UVB induction (Day 0; 20 weeks old), as the pathologic features of CLE are present after 50 days. The therapeutic effects of MMF treatment in NZBW lupus mice were examined by comparing histopathological changes, lupus band test (deposition of immune complexes at the dermal–epidermal junction) and colocalization of autoantibodies with a dermal autoantigen Dsg3, and by evaluating the associations of local matrix metalloprotease activities. Results: MMF improved survival in the NZBW lupus mice from 35.7% to 81.8%. The proteinuria, blood urea nitrogen, and interleukin 6 levels were significantly reduced after MMF treatment. The dermal lymphocytic infiltration, deposition of immune complexes at the dermal–epidermal junction, colocalized autoantibodies with Dsg3, and epidermal matrix metalloprotease activity were also attenuated in MMF‐treated NZBW F1 mice. Conclusion: The results confirmed that MMF could substantially attenuate skin damage due to CLE in the NZBW F1 mouse model.

Early activation of myocardial matrix metalloproteinases and degradation of cardiac troponin I after experimental subarachnoid hemorrhage.

BACKGROUND Matrix metalloproteinases (MMPs) are involved in acute myocardial dysfunction by degrading several intracellular contractile proteins, including cardiac troponin I (cTnI). Here, we examined the temporal profiles of MMPs and cTnI in plasma and myocardial tissue in the acute stage of subarachnoid hemorrhage (SAH). MATERIALS AND METHODS SAH was induced by the endovascular suture method in rats. Intracranial pressure and left ventricular (LV) function were recorded. Plasma cTnI and MMPs were measured at 0, 5, 15, 30, 60, 120, and 180 minutes after SAH. Myocardial cTnI and MMP activities were quantified at 30, 60 and 180 min after SAH from homogenized hearts. RESULTS SAH-induced rats showed a marked decline in -LV dP/dt(max) (index of LV diastolic function). Plasma samples revealed a noticeable increase in cTnI and pro-MMP-9 activities over the course of 180 minutes. In myocardial tissue, there was a marked increase in pro-MMP-9, pro-MMP-2 activities and expression of activated MMP-2. Western blot analysis revealed a striking decrease in cTnI content and increase in cTnI degradation in myocardium. Simultaneous cTnI depletion and MMP-2 expression in myocardium was detected by immunohistochemistry as early as 30 minutes after SAH. MMPs correlated with -LV dP/dt(max) (% of baseline) both in plasma and in myocardial tissue. Furthermore, activated MMP-2 activity correlated positively with cTnI degradation in myocardium. CONCLUSIONS Early activation of MMPs was observed in myocardium and plasma following SAH. Activated MMP-2 may regulate proteolytic cTnI and contribute to myocardium stunning injury in SAH rats.