2.1 Cultured endothelial cells
Human coronary arterial endothelial cells (HCAEC) purchased from Clonetics were seeded and incubated in endothelial cell growth medium (EGM-2-MV; Clonetics). When 70-80% confluence was achieved, cells were subcultured and plated onto 10 × 20 mm coverslips to the same confluency for immunofluorescence.
HCAECs were incubated with culture medium containing 100 nM Mito Tracker Red at 37 °C for 30 min. After washing, cells were fixed with 3.5% paraformaldehyde for 10 min. Cells were rinsed with phosphate buffered saline (PBS) and incubated with 0.1% triton X-100 for 30 min. After three 5-min washings, cells were blocked with 5% BSA, then incubated for 2 h with phallacidin (10 U/ml) or α-, β-tubulin antibodies (1:200 dilution) for labeling F-actin or microtubules, respectively, followed by wash. Secondary antibodies, fluorescein goat anti-mouse for α-, β-tubulin were applied for 1 h. Nuclei were stained with DAPI (3 μmol/l) for 30-min. After rinsing with PBS, slides were coverslipped. Mitochondria, F-actin or microtubules were detected under fluorescence microscopy with excitation/emission wavelength of 579/599 nm for Mito Tracker Red, and 492/520 nm for phallacidin or fluorescein, and 358/461 nm for DAPI labeling. The yellow fluorescence produced by overlapping red and green fluorescence identified either co-localization or superimposition of mitochondria with F-actin or microtubules. All fluorescence probes and antibodies were purchased from Molecular Probes.
2.3 Preparation of coronary arterioles
Human coronary arterioles (HCAs) were isolated from the right atrial appendage of subjects undergoing cardiac surgery as described previously [30
]. HCAs were dissected from the endocardial surface of the appendage and prepared for histofluorescence and videomicroscopy. Protocols were approved by the Medical College of Wisconsin Institutional Review Board. The investigation conformed with the principles outlined in the Declaration of Helsinki.
2.4 Detection of F-actin and microtubules in human coronary arteriolar endothelial layer
HCAs with and without 20-min intraluminal pre-incubation with cytochalasin D or nocodazole were exposed to no flow or flow for 5 min (time at which FMD achieves a steady state value) followed by 5 min fixation with 3.5% paraformaldehyde. Vessels were then opened lengthwise and placed on glass slides exposing the luminal surface. After washing, HCAs were incubated with 0.5% triton X-100 and blocked with 1% BSA. Phallotoxin 586 (5 U/ml), or α-, β-tubulin antibodies (1:200 dilution) was applied to the vessel for detecting F-actin or microtubules, respectively. Endothelium was identified by co-incubation with antibodies to Von Willebrand Factor (vWF, 1:150 dilution). Vessel segments were exposed to antibodies overnight at 4 °C. After washing, secondary antibodies (Alexa fluor 488 goat anti-rabbit IgG for VWF and Alexa fluor 594 goat anti-mouse IgG for tubulin) were applied for 30 min followed by wash. The endothelial cytoskeleton was examined using confocal microscopy with excitation wavelength of 579 nm and emission of 599 nm for detecting F-actin and microtubules, 488 and 516 nm for detecting vWF.
2.5 Fluorescence detection of flow-induced hydrogen peroxide and superoxide generation in HCAs
Dichlorodihydrofluorescein (DCFH) [31
] and hydroethidine (HE) [31
] were used to evaluate vascular production of H2
and O2 •-
, respectively, during flow. Four HCAs from the same atria were exposed to either no flow (pressurized at 60 mmHg, 0 gradient), or flow at a pressure gradient of 100 cm H2
O, in the absence or presence of intraluminal perfusion of cytochalasin D (5 lmol/l) or nocodazole (1 μmol/l). One vessel (not pressurized) treated with PEG-CAT (500 U/ml) and superoxide dismutase (150 U/ml), served as control for non-specific fluorescence. DCFH (5 μmol/l) and HE (5 μmol/l) were added in a light-protected chamber for 30 min at 37 °C either during flow or static conditions. Vessel segments were then washed with fresh PSS solution, and removed for fluorescence microscopy. DCFH and Oxy-ethidium (oxy-Etd, the fluorescent product of the reaction of HE with O2 •-
) were excited at 488 and 585 nm, and emission was measured at 526 and 620 nm, respectively. Control and experimental tissues were examined in parallel and images recorded using the same computer-specified gain and intensity settings. Images were analyzed for intensity of fluorescence within a user-defined region of the arteriolar segment (maximal traceable area of the central portion of the vessel). Artifactual autofluorescent regions were manually eliminated from analysis. Relative average fluorescence intensity was normalized for surface area and compared between control and experimental vessels.
2.6 Detection of mitochondrial superoxide production in HCA endothelium
Mitochondrial O2 •- generation in the presence or absence of flow was examined by MitoSox (5 μmol/l), a specific fluorescence probe for detecting mitochondrial O2 •- production. MitoSox was perfused intraluminally for 15 min during flow. After washing, HCAs were coverslipped and examined with confocal microscopy at excitation/emission of 510/580 nm according to the instruction from the manufacturer (Molecular Probes).
HCAs (100-150 lm) were mounted onto micropipettes for measurement of diameter [31
]. Vessels were constricted by 30-50% by adding endothelin-1 (ET-1, 10-10
to 5 × 10-10
mol/l) when spontaneous myogenic tone was not sufficient to achieve the target reduction in diameter. Flow was produced by changing the heights of the cannulating reservoirs in equal and opposite directions to generate a pressure gradient of either 20 or 100 cm H2
] in the absence or presence (20-min incubation) of inhibitors (cytochalasin D and nocodazole) or stabilizers (jasplakinolide, 100 nmol/l and taxol, 10 μmol/l) of cyto-skeleton elements. This pressure gradient is associated with steady-state flows of approximately 6 dyn/cm2
and 34 dyn/cm2
, respectively [35
]. After incubation, HCAs were re-constricted with endothelin-1 to a diameter similar to baseline diameter (without cytoskeleton inhibitors or stabilizers). A second flow-response curve was generated assessing percent dilation after each increment in flow. The responses to bradykinin (10-10
mol/l), an endothelium-dependent vasodilator and to papaverine (10-8
M), an endothelium-independent vasodilator that acts via a mechanism distinct from shear stress [2
], were recorded in some vessels to assess the specificity of the effects of cytochalasin D and nocodazole.
All data are expressed as mean ± SEM. The relative fluorescence intensities observed in arterioles exposed to static conditions or flow were compared using a two way ANOVA. Percent dilation was calculated as the percent change from preconstricted diameter to the diameter after flow or agonist (normalized to the maximal diameter measured after papaverine, 10-4 mol/l). Data from vessels exposed to flow before and after treatment with cytoskeleton antagonists were compared using a one-way ANOVA. All differences were judged to be significant at the level of p < 0.05.