Beneficial Effects of Necrosis Modulator, Indole Derivative NecroX-7, on Renal Ischemia-Reperfusion Injury in Rats.

Renal ischemia-reperfusion injury (IRI) is involved in multiple diseases, such as kidney transplantation or contrast-induced nephropathy, and leads to acute kidney injury. However, there are no pharmacological agents available to prevent IRI. In this study, we investigated the effects of necroX-7 against renal IRI in a rat model. Seven-week-old male Sprague-Dawley rats were divided into four groups: saline-treated sham or IRI group, necroX-7-treated sham or IRI group. All animals had right nephrectomy and IRI was followed by reperfusion after clamping the left renal vessels for 35 minutes. NecroX-7 or saline was intravenously injected at 5 minutes before reperfusion. The effects of necroX-7 on IRI were evaluated using biochemical, histological, and molecular markers. The serum creatinine level was increased after IRI compared with sham. The necroX-7 significantly decreased creatinine level compared with the saline in IRI (1.36 ± 0.11 vs 2.35 ± 0.42 mg/dL; P < .05). An immunohistochemical study revealed that necroX-7 improved renal tubular injury, and attenuated 8-OHdG-positive cells (P < .001) and high-mobility group Box 1 protein (HMGB1) expression compared with saline treatment in IRI (P < .001). NecroX-7 significantly reduced monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß in IRI (necroX-7-treated IRI vs saline-treated IRI rats; 1.73 ± 0.42 vs 7.23 ± 0.54-fold for MCP-1, P < .05; 0.79 ± 0.59 vs 3.72 ± 0.37-fold for TNF-α, P < .05; 0.50 ± 0.36 vs 2.43 ± 0.41-fold for IL-1ß, P < .001). In conclusion, necroX-7 improved renal dysfunction after IRI. These effects of necroX-7 occurred with the suppression of reactive oxygen species, HMGB1, and inflammatory responses. We suggest that necroX-7 has potential therapeutic benefits in renal IRI.

Activation of CD14 on circulating monocytes in patients with acute coronary syndrome.

BACKGROUND: Increasing evidence supports the involvement of inflammation in acute phase of coronary artery diseases. METHODS: We analyzed the status of activation of inflammatory cells in 38 patients with acute coronary syndrome, 14 stable angina patients, and 19 control subjects by flow-cytometry. Expression levels of CD14 and the percentage of HLA-DR(+) T-lymphocytes were used as markers of monocyte and T-lymphocytes activation, respectively. RESULTS: The expression of CD14 on monocytes in acute coronary syndrome patients (mean fluorescence intensity+/-S.D.=158.1+/-77.1) was increased significantly in comparison to control subjects (57.1+/-8.0) and the stable angina group (63.6+/-22.0) (P<0.0001 for both). A significantly higher percentage of HLA-DR positive T-lymphocytes (20.4+/-9.0 vs. 12.7+/-3.7%, P<0.01) was observed in acute coronary syndrome patients in comparison to control subjects. Incubation of whole blood cells with bacterial lipopolysaccharide resulted in a 2.4-fold higher secretion of tumor necrosis factor-alpha in acute coronary syndrome patients than in control subjects (P<0.05). When these markers of activation were measured in acute coronary syndrome patients 6 weeks after medical treatment, a significant reduction both in monocytic CD14 expression and percentage of HLA-DR positive T-lymphocytes (P<0.05 for both) was observed. DISCUSSION: We observed markedly increased levels of monocytic CD14 expression in ACS patients, which appear to indicate the activated status of monocytes and hyper-responsiveness to external stimuli. The CD14 expression levels decreased as the patients were treated, indicating that the expression of CD14 accurately represents the activation status of monocytes during the acute phase of coronary artery diseases.

The feasibility and safety of fluoroscopy-guided percutaneous intramyocardial gene injection in porcine heart.

BACKGROUND: Catheter-based transendocardial gene injection would be useful for the delivery of genes into the heart. We examined the feasibility and safety of percutaneous intramyocardial gene injections with fluoroscopic guidance alone. METHODS: We performed the procedure through an 8F arterial sheath inserted into the left carotid artery. In protocol 1, a mixture of India ink and normal saline was injected through a needle injection catheter in six pigs. We monitored blood pressure and ECG continuously during the procedure. Echocardiography, left ventriculography, and coronary angiography were performed. All pigs were sacrificed 2 days later and hearts were harvested. In protocol 2, a mixture of India ink and plasmid encoding CAT gene was injected in the same manner in eight pigs. Myocardial tissue was obtained 7 days after the procedure to assess gene expression. In protocol 3, four pigs were intentionally needle-perforated in the ventricular wall and were observed for 7 days. RESULTS: In protocol 1, there was no significant hemodynamic changes or serious arrhythmias during the procedure. Echocardiography and angiography revealed no evidence indicating pericardial effusion or wall motion abnormalities. Harvested hearts revealed one intramyocardial hematoma in a total of 36 injection sites. In protocol 2, the gene expression could be identified in 39 sites out of 48 injections after 7 days. In protocol 3, no animal showed signs indicating cardiac tamponade during the observation period. CONCLUSIONS: Our data suggest that fluoroscopy-guided percutaneous intramyocardial gene injection is a feasible and safe procedure, with no indication of associated significant hemodynamic changes, arrhythmias, or mortality.

Intracardiac echocardiographic guidance and monitoring during percutaneous endomyocardial gene injection in porcine heart.

In an effort to develop a guiding and monitoring tool for transmyocardial gene transfer, we have evaluated the feasibility of intracardiac echocardiography (ICE) to guide percutaneous endomyocardial gene transfer (PEGT), and monitor complications, in a pig model. ICE (5.5-10 MHz), complemented by fluoroscopy, was utilized to guide a needle injection into the heart in 19 normal pigs. Using this system, we injected Evans blue dye into eight pigs (group I), a mixture of pCK-CAT plasmid and India ink into seven pigs (group II), and pCK-LacZ plasmid into four pigs (group III). In all pigs, ICE contributed to the injection procedure by guiding the catheter to anatomically distinct sites, and by assisting stabilization of the catheter-endocardial contact. ICE predicted the injection sites correctly in 56 of 64 sites (87.5%) in group I, and in 42 of 42 sites (100%) in group II. Leakage of injectate into the left ventricular cavity could be detected by the microbubbles generated. The sites of injections appeared as foci of bright myocardial echodensity, which persisted until the end of the procedure. The procedures were not associated with significant morbidity or mortality. The expression of the chloramphenicol acetyltransferase (CAT) gene was identified in 40 sites from 42 injections (95.2%) in group II. In group III, histology showed positive beta-galactosidase staining of myocytes limited around the needle track with low transfection efficiency (<1%). These results suggest that real-time ICE monitoring proves safe and useful during PEGT for guiding needle injection, monitoring leakage, ensuring delivery of injectate into the myocardium, and instantly diagnosing cardiac complications, resulting in successful gene transfer.

Clinical features and prognosis of acute aortic intramural hemorrhage compared with those of acute aortic dissection: a single center experience.

The clinical manifestations and natural history of acute aortic intramural hemorrhage are not well characterized. Therefore, we have evaluated the differences in the clinical features and prognosis between acute intramural hemorrhage and acute classic aortic dissection. One hundred two consecutive patients with acute aortic syndrome were diagnosed between November 1994 and May 1999. The clinical features, treatment modalities and survival of these patients were analyzed. Thirty one of the 102 patients (30%) had intramural hemorrhage and 71 (70%) had aortic dissection. Patients with intramural hemorrhage were older than those with aortic dissection (mean ages 67 and 55 years, respectively) (p < 0.001), and intramural hemorrhage showed a lower proportion of type A than did aortic dissection (32% and 58%, respectively) (p = 0.018). The incidence of severe complications was significantly lower in patients with intramural hemorrhage than in those with aortic dissection (19% and 27%, respectively) (p < 0.001). Mean follow-up duration was 23.1+/-16.0 months. The overall death rate for patients with intramural hemorrhage (2 / 31; 6%) tended to be lower than those with aortic dissection (14 / 71; 20%) (p = 0.104). The Stanford classification and treatment modalities were not correlated with death. Late follow-up imaging studies in intramural hemorrhage showed partial to complete resolution of intramural hematoma (9 / 15; 60%). In this study, intramural hemorrhage was fairly common, more frequent among older patients, had a lower proportion of type A, and showed a lower incidence of severe complications and a more favorable prognosis in terms of mortality, than aortic dissection.

Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector.

Vascular endothelial growth factor (VEGF) has proven to be one of the most effective growth factors for therapeutic angiogenesis. The biological efficacy of the adeno-associated virus (AAV) vector has recently been demonstrated in muscle tissues, including the heart. Apart from these promising insights into VEGF and the AAV vector, studies on VEGF gene transfer using the AAV vector have been limited. Here, we evaluate AAV-mediated VEGF gene transfer, both in vitro and in vivo, using the AAV-mVEGF vector that contains cDNA for murine VEGF(120) within an HCMV-driven expression cassette. Transient transfection of AAV-mVEGF plasmid significantly increased mVEGF expression in 293T cells. The secreted VEGF in the conditioned medium had strong biological activity, as confirmed by the Miles' vascular permeability assay. Transduction of 293T and HeLa cells with AAV-mVEGF stock of high titer, that is essentially adenovirus-free, showed significantly increased mVEGF expression above that of AAV-eGFP-transduced cells. When human umbilical vein endothelial cells were transduced, a higher level of mVEGF expression, together with higher cell counts, was observed compared to AAV-eGFP-transduced cells. In vivo transduction of mouse tibialis anterior muscle resulted in an increased level of mVEGF expression, and higher capillary-to-myofibre ratio, 8 weeks post-transduction. In a rat hindlimb ischemia model, regional blood flow, as well as the capillary-to-myofibre ratio, was significantly increased at 4 weeks post-transduction. These findings demonstrate the efficient delivery of the VEGF gene using an AAV vector, which has implications for angiogenic gene therapy in ischemic diseases.