Lovastatin

The antimicrobial potential of lovastatin produced by A. terreus AUMC 15760, compared to the standard lovastatin (Sigma-Aldrich), was tested against three fungi (C. albicans AUMC 13415, C. glabrata AUMC 13412, and C. krusei AUMC 13420) and three strains of E. coli ATCC 8739, S. aureus ATCC 6538, and S. epidermidis ATCC 12228. Both drugs were used at concentrations of 5, 2.5, 1.25, 0.62, and 0.31 mg/mL. Nystatin (50 mcg/disc) and Piperacillin/Tazobactam 10:1 (110 µg/disc) were used as antifungal and antibacterial reference drugs, respectively. The test organisms were grown in 24-h-old cultures, and spore suspensions (10⁸ CFU/mL = 0.5 McFarland standard solution) were employed to inoculate Petri plates. Using sterile cotton swabs and a sterile Petri plate filled with either Sabouraud’s dextrose agar (SDA) for Candida species or nutritional agar (NA) for bacteria, the bacterial and fungal spore suspensions were evenly distributed. Using the well diffusion method [109 (link)], the five wells (5 mm-diameter holes cut in the agar gel) each received a 50 µL addition of the tested drug. The plates were incubated for 24 h at 37 ± 1 °C for bacteria and Candida under aerobic conditions. After incubation, the inhibition of bacterial and fungal growth was measured in mm. Tests were performed in triplicate.

Samples of normal hamstring tendon were obtained from patients, average age 30 (19–45y), undergoing anterior cruciate ligament reconstruction with informed consent and in full compliance with UK HTA and MREC requirements and with the Helsinki Declaration of 1975, as revised in 1983. Tenocytes were derived by standard explant protocol (Torricelli et al., 2006). They were expanded in DMEM/F12 (Lonza, Cheltenham, UK) containing 10% foetal calf serum (FCS) (Biosera, Ringmer, UK) and maintained in DMEM/F12 5% FCS during statin treatment. Tenocytes were treated with 0.2–2 μM lovastatin (Sigma, Poole, UK) dissolved in dimethyl sulfoxide (DMSO) (Sigma) for different times. Furthermore the cells were incubated in the presence of 0.08 and 0.8 μM atorvastatin (Sigma) and 0.048 and 0.48 μM simvastatin (Sigma). The latter was dissolved before treatment in NaOH in ethanol. To reverse lovastatin‐induced changes 100 μM mevalonate (Sigma) was added back. Vehicle controls included DMSO for lovastatin and atorvastatin, NaOH in ethanol for simvastatin and ethanol for mevalonate. Cells were used at passage 4 or below as it has been reported that tenocytes may dedifferentiate at higher passages (Yao et al., 2006).

N2a cells and astrocytes were depleted of cholesterol by two alternative methods: MβCD treatment to extract cholesterol from the membrane and lovastatin to inhibit its synthesis. For MβCD (Sigma, Saint Louis, MO, USA; cat#C4555) treatment, cells were washed with PBS and incubated with 5 or 10 mM MβCD in serum free DMEM for 60 min at 37 °C. Cholesterol enrichment or repletion of cholesterol-depleted cells were obtained by incubation of the cells for 2 h at 37 °C using MβCD-cholesterol inclusion complexes (formed by mixing cholesterol suspension with cyclodextrin solution, as described [47 (link)]). To decrease cholesterol synthesis, we applied lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the enzyme that catalyzes the conversion of HMH-CoA to the cholesterol precursor mevalonate. For studies with lovastatin, the cells were incubated 12 h with 1, 5, or 20 µM lovastatin (Sigma) in serum free DMEM at 37 °C.

Lovastatin (Sigma, St. Louis, MO, United States) was dissolved in DMSO to prepare a stock solution of 6 mM. Cells were treated with different concentrations of Lovastatin for 4 h and then transduced with α-syn PFFs. Lovastatin was maintained in the medium. The cells were harvested 48 h after transfection.

Endothelial dysfunction and recovery was evaluated via acute exposure to TNF-α59 (link). Endothelialized TEBVs were allowed to mature for 7 days. A 500 μL media sample was collected and frozen at −20 °C for NO analysis. TEBV vasoactivity was evaluated after 7 days using the phenylephrine-acetylcholine assay listed above. TNF-α was introduced to the flow circuit for 4.5 hours at a final concentration of 200 U/mL. TEBV vasoactivity was evaluated after 7 days using the phenylephrine-acetylcholine assay. Media was changed directly afterward to TEBV media with 2 mg/mL ε-aminocaproic acid. TEBV vasoactivity was assessed using the phenylephrine-acetylcholine assay three days and seven days later to evaluate endothelial recovery after removal of the TNF-α stimulus.
The effect of lovastatin toward mediating TEBV response toward TNF-α was evaluated by culturing endothelialized TEBVs made with hNDFs for 1 week. On Day 7, lovastatin (Sigma) was introduced into the flow circuit to produce a final concentration of 1 μM. Control TEBVs were evaluated without exposure to lovastatin. TEBVs were treated for 3 days, and then exposed to 200 U/mL TNF-α for 4.5 hours. TEBV vasoactivity was assessed using the phenylephrine-acetylcholine assay at Day 7, Day 10, and Day 10 after treatment with TNF-α.

A lovastatin (Sigma-Aldrich, St. Louis, MO, USA) stock solution at a concentration of 0.124 M was prepared by dissolving in DMSO and applied at a final concentration of 123.6 μM. A solution of 20 μL lovastatin and 1 μL Tween-20 was applied on the cut surfaces of 10 tomato stems after decapitation. An additional equal number of tomato plants were treated with water as control. The number of regenerated adventitious buds was counted on 0, 30, 37, 44, 51 and 58 d after decapitation, respectively. We used ANOVA to test the difference between the treatment with and without lovastatin added.