Journal: Respiratory Research

Article Title: Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

doi: 10.1186/1465-9921-12-103

Figure Lengend Snippet: Increased expression of PDGF-BB in pulmonary endothelial cells on treatment with HIV-proteins . Representative western blot showing PDGF-BB expression in cellular extracts from Tat (25 ng/ml), gp-120 LAV (100 ng/ml), gp-120 CM (100 ng/ml), heat-inactivated (HI) Tat or HI-gp-120 treated human pulmonary microvascular endothelial cells. The blots were re-probed with human β-actin antibodies. Histogram represents the average densitometric ratio of PDGF-BB to β-actin of three independent experiments. Statistical significance was calculated using a two-tail, independent t-test. (** p ≤ 0.01 vs. control, #p ≤ 0.05 vs. Tat or gp-120 treatment).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin.

Techniques: Expressing, Western Blot, Control

Journal: Respiratory Research

Article Title: Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

doi: 10.1186/1465-9921-12-103

Figure Lengend Snippet: Involvement of oxidative stress in gp-120 mediated PDGF-BB induction in pulmonary endothelial cells . A) Enhanced oxidative stress in pulmonary endothelial cells on Tat and gp120 treatment. Human pulmonary microvascular endothelial cells (HPMVECs) were incubated with carboxy-H2-DCF-DA followed by Tat (25 ng/ml) or gp-120 (100 ng/ml) treatment for 60 min, and assessed for oxidative stress (Mean ± SD., **P ≤ 0.01, ***P < 0.001 vs. control). B) Effect of CCR5 neutralizing antibody on gp-120 CM (100 ng/ml) mediated oxidative stress in HPMVECs. Cells were pretreated with CCR5 antibody (10 μg/ml) or equal amount of IgG isotype control for 30 min, followed by DCF assay (Mean ± SD., ***P < 0.001 treatment versus control; #P < 0.05 vs. gp120 CM treatment). C) Gp-120 CM mediated PDGF-BB expression in the presence of antioxidant cocktail. HPMVECs were pretreated with antioxidant cocktail for 30 min followed by incubation with gp-120 CM (100 ng/ml) for 24 hours. Cells were then used for protein extraction followed by sequential immunobloting with antibodies specifically directed to the PDGF-BB and β-actin. Representative western blot images (upper panel) are shown with histograms (lower panel) showing the average densitometric analysis of the PDGF-BB band normalized to corresponding β-actin band from three independent experiments (*** P < = 0.001 versus control; ###P < = 0.001 versus gp120 CM treatment).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin.

Techniques: Incubation, Control, DCF Assay, Expressing, Protein Extraction, Western Blot

Journal: Respiratory Research

Article Title: Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

doi: 10.1186/1465-9921-12-103

Figure Lengend Snippet: Oxidative stress dependent HIF-1α expression is involved in gp-120 CM mediated PDGF-BB induction . A) Western blot analysis of HIF-1α expression in human pulmonary microvascular endothelial cells (HPMVECs) pretreated with or without antioxidant cocktail for 30 min followed by incubation with gp-120 CM (100 ng/ml) for 24 hours. B) Evaluation of HIF-1α knockdown by western blot analysis of whole cell lysates from HPMVECs transfected with siRNA specific to HIF-1α (10nM) or with negative control siRNA in presence of gp120 CM treatment. ( C) Knock down of HIF-1α resulted in inhibition of gp120 CM -mediated induction of PDGF-BB expression in HPMVECs. Blots are representative of three independent experiments with histogram (lower panel) showing the average densitometric analysis normalized to β-actin. All values are mean ± SD. *P < = 0.01,**P < = 0.001 treatment versus control; #P < = 0.01, ##P < = 0.001 treatment versus gp120 CM treated untransfected cells.

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin.

Techniques: Expressing, Western Blot, Incubation, Knockdown, Transfection, Negative Control, Inhibition, Control

Journal: BMC Microbiology

Article Title: Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

doi: 10.1186/1471-2180-12-7

Figure Lengend Snippet: Invasion and survival of M. tuberculosis pknD mutant in host-derived cells . A . BALB/c mice were infected with M. tuberculosis CDC1551 or pknD mutant, and sacrificed at days 1 and 49 after infection. The mutant for M. tuberculosis pknD was significantly attenuated (P = 0.004) in mouse brain, but not lung tissue, 49 days after infection. No defect was observed in the lungs at either time point. Bacterial burden is represented as log 10 CFU/organ for all animal experiments. B . Invasion of host-cell monolayers by wild-type CDC1551, wild-type intergenic transposon control, pknD transposon mutant (pknD:Tn), and pknD genetic complement (pknD:Comp) was examined and normalized to the wild-type control. Invasion assays were performed in brain microvascular endothelial cells (HBMEC), epithelial A549 cells, and umbilical vein endothelia (HUVEC). No difference in invasion was observed in A549 cells (P = 0.31) or HUVEC (P = 0.41). A significant reduction in invasive capacity, however, was observed in the CNS-derived HBMEC (P = 0.02). This defect was restored by genetic complementation with the native pknD/pstS2 operon. N.S. = not significantly different. C . Intracellular survival of each of the above M. tuberculosis strains was examined in HBMEC at days 1, 3, 5, and 7 after infection. The pknD:Tn mutant demonstrated an invasion and intracellular survival defect in HBMEC relative to wild-type over the course of the seven day infection. D . Survival was also examined by infection of activated J774 macrophages. No corresponding survival defect for the pknD:Tn mutant was observed in these cells during the seven day infection. A mutant for the gene Rv0442c , known to be attenuated in the macrophage model, is included as a control. All CFU counts are represented as mean ± standard deviation.

Article Snippet: Primary human brain microvascular endothelial cells and HUVEC were kind gifts from Dr. Kwang Sik Kim, Department of Pediatrics, Johns Hopkins University School of Medicine.

Techniques: Mutagenesis, Derivative Assay, Infection, Control, Standard Deviation

Journal: Diabetes

Article Title: Decreased Cerebrovascular Brain-Derived Neurotrophic Factor–Mediated Neuroprotection in the Diabetic Brain

doi: 10.2337/db10-1371

Figure Lengend Snippet: BDNF expression is reduced in the diabetic brain endothelium. Representative images of cortical sections (2-mm thick) from 6-week diabetic and age-matched nondiabetic rats immunostained for BDNF. Blood vessels (arrows) are visualized by CD31 staining ( left ). Cortex microvessels from a diabetic rat show decreased BDNF immunofluorescence when compared with those of a nondiabetic rat ( right ). Magnification 40×. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Primary human brain microvessel endothelial cells were purchased from Cell Systems Corporation (Kirkland, WA), mostly derived from a heterogenous mix of rapidly autopsied human brains obtained within a few hours after death.

Techniques: Expressing, Staining, Immunofluorescence