Decreasing mitochondrial fission diminishes vascular smooth muscle cell migration and ameliorates intimal hyperplasia.

AIMS: Vascular smooth muscle cell (VSMC) migration in response to arterial wall injury is a critical process in the development of intimal hyperplasia. Cell migration is an energy-demanding process that is predicted to require mitochondrial function. Mitochondria are morphologically dynamic, undergoing continuous shape change through fission and fusion. However, the role of mitochondrial morphology in VSMC migration is not well understood. The aim of the study is to understand how mitochondrial fission contributes to VSMC migration and provides its in vivo relevance in the mouse model of intimal hyperplasia. METHODS AND RESULTS: In primary mouse VSMCs, the chemoattractant PDGF induced mitochondrial shortening through the mitochondrial fission protein dynamin-like protein 1 (DLP1)/Drp1. Perturbation of mitochondrial fission by expressing the dominant-negative mutant DLP1-K38A or by DLP1 silencing greatly decreased PDGF-induced lamellipodia formation and VSMC migration, indicating that mitochondrial fission is an important process in VSMC migration. PDGF induced an augmentation of mitochondrial energetics as well as ROS production, both of which were found to be necessary for VSMC migration. Mechanistically, the inhibition of mitochondrial fission induced an increase of mitochondrial inner membrane proton leak in VSMCs, abrogating the PDGF-induced energetic enhancement and an ROS increase. In an in vivo model of intimal hyperplasia, transgenic mice expressing DLP1-K38A displayed markedly reduced ROS levels and neointima formation in response to femoral artery wire injury. CONCLUSIONS: Mitochondrial fission is an integral process in cell migration, and controlling mitochondrial fission can limit VSMC migration and the pathological intimal hyperplasia by altering mitochondrial energetics and ROS levels.

Decreasing mitochondrial fission prevents cholestatic liver injury.

Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.

A new approach to ELISA-based anti-glycolipid antibody evaluation of highly adhesive serum samples.

The enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay used in measuring antibody reactivity (expressed as titers) for glycosphingolipids (GSLs) such as gangliosides and sulfoglycolipids in the sera of patients with Guillain-Barré syndrome (GBS), variants of GBS, and chronic inflammatory demyelinating polyneuropathy (CIDP). In the present study, anti-GSL antibodies were evaluated using a new formula of affinity parametric complex (APC), calculated from limiting-dilution serum assay data, followed by affinity parametric complex criterion (APCC). Using assay results based on APCC, we analyzed serum samples categorized into acute inflammatory demyelinating polyneuropathy (AIDP), acute motor-sensory axonal neuropathy (AMSAN), CIDP, CIDP with myasthenia gravis (MG), and amyotrophic lateral sclerosis (ALS). We were able to determine the affinity strength of antibodies otherwise hidden in the non-specific background activity in highly adhesive serum samples. The thin-layer chromatography (TLC)-immuno-overlay method assured us that this new method is an accurate and reliable way for evaluating anti-GSL antibodies using ELISA serum sample data.

Thin-layer Pap test vs. conventional Pap smear. Analysis of 400 split samples.

OBJECTIVE: To analyze our experience with 400 Thin-Prep (TP) split samples (Cytyc Corp., Boxborough, Massachusetts) as an initial assessment of this new technology's effect in our laboratory. STUDY DESIGN: Three gynecologic oncologists and two general gynecologists obtained the 400 split samples using a broom sampling device. Following conventional smear (CS) preparation, they rinsed the broom in Preservcyt solution (Cytyc) for subsequent TP processing. The paired samples were separated, independently analyzed and classified by the Bethesda System. All available follow-up surgical pathology material was reviewed and compared to the cytologic diagnoses. RESULTS: TP had significantly more abnormal results (22% vs. 16%, P = .007), including more atypical squamous cells of undetermined significance (ASCUS) (9.5% vs. 6.3% P = .07) and low grade squamous intraepithelial lesion (LSIL) (7.8% vs. 5.3%, P = .03). Both methods had 3.3% high grade squamous intraepithelial lesion (HSIL). For TP, ASCUS/squamous intraepithelial lesion (SIL) = 0.86 and for CS, ASCUS/SIL = 0.74. Ten TP SILs had a paired negative CS, including LSIL (nine cases) and HSIL (one case). Consensus review of these 10 TP slides confirmed the HSIL and four LSILs. No CS SILs had a paired negative TP. Only 36 (9%) cases had surgical pathology follow-up. The surgical specimens included 17 cervical intraepithelial neoplasia (CIN) 2 or above. The TP method had no false negatives, while the CS method had 3 false negatives among the 17 confirmed cases of CIN 2 or above. CONCLUSION: TP appears to be superior to CS for detecting SILs.