7
MS#1087/R1 Suppl.
EXPANDED MATERIALS AND METHODS
Solutions and Chemicals
An albumin-physiological salt solution (APSS) was used as a bathing solution while the microvessels were being dissected. It contained the following (in mM): NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and 3-N-morpholino propanesulfonic acid buffer 3.0. After the addition of 1% bovine serum albumin, the solution was buffered to a pH of 7.40 at 4°C and then filtered through a Millex-PF 0.8 µm filter unit (Millipore, Bedford, MA). The APSS used to perfuse the vessels during permeability measurements had the same composition as mentioned above, but was buffered to a pH of 7.40 at 37°C. The chemicals used to make the perfusate, including FITC-albumin, were purchased from Sigma (St. Louis, MO). Bovine serum albumin was obtained from United States Biochemical (Cleveland, OH). The PKC inhibitors, bisindolylmaleimide (BIM), Goe 6976, hispidin, and HBDDE were obtained from Calbiochem (San Diego, CA). Streptozotocin (STZ) was obtained from Upjohn (Dallas, TX). Antibodies to PKCa, PKCbI, and PKCbII and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz (Santa Cruz, CA), and anti-PKCe was from Transduction Laboratory (Lexington, KY).
Diabetic Animal Model
Diabetes was induced in juvenile pigs by selective ablation of the pancreatic b-cell with intravenous injection of streptozotocin. Yorkshire pigs weighing 9-12 kg were fed with a commercial pig diet containing ammonium chloride (20 g/kg hog chow) the day prior to STZ injection, producing temporary systemic acidosis and thus enhancing the effect of STZ. STZ was freshly prepared and injected through a central vein of sedated pigs at 150 mg/kg. The pigs were allowed to live for 4-8 weeks with free access to water and commercial diet. The level of blood glucose and pH were closely monitored daily. Only those developing sustained hyperglycemia with a blood glucose level above 300 mg/dL (about 17 mM) were included in this study. The average blood glucose level in pigs used in the experiments was 414 ± 26 mg/dL (23.0 ± 1.4 mM).
The pigs were housed in an Institutional Animal Care and Use Committee (IACUC) approved and supervised facility. The experimental protocols were approved by the IACUC and followed the NIH guideline for experimental animal care and use.
Isolation and Perfusion of Coronary Venules
Pigs were anesthetized with sodium pentobarbital (25 mg/kg, iv) and heparinized (250 units/kg, iv). Following a tracheotomy and intubation, the animal was ventilated. A left thoracotomy was performed and the heart was electrically fibrillated, excised and placed in 4°C physiological saline. The coronary sinus was cannulated, and 3 ml India ink-gelatin-physiological salt solution was infused to clearly define venular microvessels. This solution was prepared by adding 0.2 ml of India ink (Koh-I-Noor, Bloomsbury, NJ) and 0.35 g of porcine skin gelatin to 10 ml of warm physiological salt solution and filtered through P8 filter paper (Fisher Scientific, Pittsburgh, PA). Information regarding the validation and limitation of the ink-perfusion procedure has been provided in our previous publications (1), which also provides the technical detail of the isolated and perfused coronary venule model. Briefly, a suitable venule (length 0.8-1.2 mm, diameter 30-50 µm) was dissected from the surrounding myocardium in a dissecting chamber containing APSS at 4°C with the aid of a Zeiss stereo dissecting microscope. The vessel was transferred to a cannulating chamber, which was mounted on a Zeiss inverted microscope. The isolated vessel was cannulated with 2 micropipettes outer pipettes and secured with 11-0 suture (Alcon, Fort Worth, TX). A smaller pipette (inner pipette) was inserted into each of the outer pipette. The pipette-in-pipette cannulation technique allowed us to perfuse the vessel interchangeably with different solutions; thus various chemicals or different concentrations of a chemical could be tested in the same preparation. Each cannulating micropipette was connected to a reservoir to allow independent control of intraluminal pressure and flow. The vessel was perfused at a constant pressure of about 10 cmH2O and a flow rate of 7 mm/sec. The bath solution in the chamber was maintained at 37°C and pH 7.4 throughout the experiments. The image of the vessel was projected onto a Hamamatsu charged coupled device-intensified camera and was displayed on a high-resolution monochromatic video monitor and recorded onto a VHS video recorder. Diameter of the vessel was measured on-line with a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX). The preparations were discarded if leaks were detected.
Measurement of Venular Permeability
The permeability of the vessel was measured with a fluorescence ratioing technique (2). Using an optical window of a video photometer positioned over the venule and adjacent space on the monitor, the fluorescent intensity from the window was measured and digitized on-line by a Power Macintosh computer. In each measurement the isolated venule was first perfused with APSS through the outer inflow pipette to establish baseline intensity. The venular lumen was then rapidly filled with fluorochromes by switching the perfusion to the inner inflow pipette which contained FITC-albumin. This produced an initial step-increase, followed by a gradual increase, in the intensity of fluorescence. There was a step-decrease of intensity when the fluorescent-labeled molecules were washed out of the vessel lumen by switching the perfusion back to the outer inflow pipette. The apparent solute permeability coefficient of albumin (Pa) was calculated using the equation Pa = (1/DIf) (dIf/dt)o (r/2), where, DIf is the initial step increase in fluorescent intensity, (dIf/dt)o is the initial rate of gradual increase in intensity as solutes diffuse out of the vessel into the extravascular space, and r is the venular radius. In previous experiments, the permeability was monitored over 6 hours to ensure that the permeability properties of the venules were not significantly altered with time (1).
Cell Culture and Treatment
Coronary venular endothelial cells were isolated from postcapillary venules as previously described (3, 4). Cells were routinely maintained on gelatin-coated dishes containing 20% fetal bovine serum (FBS) in complete DMEM (DMEM with 1 mM sodium pyruvate, 2 mM l-glutamine, 15 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 25 U/ml heparin). The cells exhibited properties characteristic of the endothelial cell, such as typical cobblestone morphology, positive immunofluorescent staining for factor VIII antigen, uptake of diacetylated low-density lipoprotein, and the ability to form tubes. To study the effect of glucose on PKC activity, the cells were incubated for 24 hours with the same culture media containing 50 mM of D-glucose. Cells grown in normal glucose (5 mM) served as controls. The cells were then lysed for enzymatic analysis of PKC activity.
Assay of Protein Kinase Activity
PKC activity was measured in cultured coronary venular endothelial cells as well as in freshly isolated coronary venules. A MESACUP protein kinase assay kit (Medical and Biological Laboratories, Nagoya, Japan) was used according to their protocol. Briefly, coronary venular endothelial cells were grown to confluence and then lysed and sonicated for 5 minutes. For the venular study, twenty to thirty coronary venules ranging from 30 to 50 mm in diameter were dissected from the pig heart and quickly homogenized in a lysis buffer. The soluble fraction of the cells was then collected by centrifugation at 16,000g for 30 min at 4°C. The supernatant was harvested and added to a PS-peptide coated 48-microwell plate for 10 min at room temperature. The sample was then incubated with a biotinylated monoclonal antibody 2B9 for 1 hr followed by POD-conjugated streptavidin for another hour at room temperature. Finally, a peroxidase substrate solution was added and the optical density of the sample was read at 492 nm with a microplate reader.
Subcellular Fractionation and Western Blot Analysis
The subcellular localization of PKC isoforms was examined by quantitative immunoblotting of membrane and cytosolic fractions of the heart tissue. Left ventricular myocardium (0.5 g) was homogenized with a tissue grinder kit (Wheaton Scientific Product, New Jersey) in lysis buffer containing 20 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 6 mM 2ME, 50 mM NaF, 1 mM PMSF, 1 mM Na3VO4, approtinin 10 µg/ml, and leupeptin 10 µg/ml. The sample was passed through 25G needles, sonicated at 4°C for 20 min, and then centrifuged at 10,000g for 3 min. The crude particulate fraction was discarded and supernatant was subjected to ultracentrifugation at 100xg for 60 min at 4°C. The supernatant was harvested as cytosolic fraction. The pellet was resuspended in homogenizing buffer containing 1% Triton X-100, sonicated at 4°C for 20 min, and centrifuged at 100xg for 60 min. The resulting detergent-extracted supernatant was collected as the membrane fraction.
For Western blot assay, equal amount of cytosolic and membranous protein extracts were separated by 4-12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked overnight in Superblock blocking buffer (Pierce, Rockford, IL) with PKC isoform-specific primary antibodies at 4°C. Blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:2500 dilution) for 45 min. Immunoreactive bands were visualized with enhanced chemiluminescence. Images of the bands were scanned by reflectance scanning densitometry and the intensity of the bands was quantified using NIH image software.
Experimental Protocols
First, the direct effect of high glucose on venular barrier function was evaluated. The apparent permeability coefficient of albumin was measured in isolated coronary venules during intraluminal perfusion of various concentrations (5, 12.5, 25, 50, and 100 mM) of D-glucose. To factor out the confounding effect of hyperosmolarity produced by high concentrations of glucose, the osmolarity of APSS was adjusted to 290-310 mmol/kg and both the perfusate and suffusate contained the same chemical ingredients.
Subsequently, the role of PKC in mediation of the injurious effect of glucose was examined by measuring the venular permeability response to high glucose during pharmacological inhibition of PKC. Two selective PKC inhibitors, BIM and Goe 6976 were used. In separate experiments, venules isolated from control pigs were first incubated under normal glucose (5 mM) with either BIM (10-5 M) or Goe 6976 (10-7 M) for 30 min, and Pa was measured to confirm that the drugs did not significantly alter the basal permeability property of the vessels. Then the vessels were subjected to a high concentration (50 mM) of glucose for 60 min and the permeability response was again measured in the presence of the PKC inhibitor.
In order to correlate the effect of glucose with that of diabetes, the permeability measurement was conducted directly on coronary venules dissected from streptozotocin-induced diabetic pigs at 2, 4, and 6-8 weeks after diabetes induction. The basal Pa values of the diabetic vessels were compared to those obtained from age-matched control pigs. To evaluate the impact of PKC inhibition on restoration of the barrier function, the diabetic vessels were incubated with the wide-spectrum PKC inhibitor BIM (10-7 - 10-5 M, 40 min) as well as the selective inhibitors of PKC a-isoforms (HBBDE, 8x10-5 M, 40 min), and b-isoform (hispidin, 4x10-6 M), respectively. Then the venular permeability was compared before and after inhibition of PKC. The PKC antagonists and their dosages were chosen based on previous literature (5, 6) and our control studies that confirmed the efficacy of the antagonists in both cultured coronary venular endothelial cells and isolated coronary venules.
Finally, the activity of PKC was directly measured in freshly isolated coronary venules of diabetic animals as well as in venular endothelial cells cultured under high glucose condition. A protein kinase assay was performed to analyze the enzymatic activity of PKC. To examine the activation status of specific PKC isoforms, Western blot analysis was performed on the cytosolic and membranous fractions of the heart tissue. The subcellular distribution of PKCa, PKCbI, PKCbII, and PKCe was compared between control and diabetic pigs.
Data Analysis
In the intact vessel studies, the Pa was measured two to three times for each venule at each experimental intervention and the values were averaged. For all experiments n is given as the number of vessels studied. At each experimental condition, the values of the Pa from different venules were averaged and reported as mean±SE. For the PKC assay, at least three samples were obtained from different dishes of cells or groups of vessels and the optical density of the samples were averaged. Analysis of variance was used to evaluate the significance of intergroup differences. A value of P<0.05 was considered significant for the comparisons.
References
1. Yuan Y, Mier RA, Ghilian WM, Granger HJ, Zawieja DC. Permeability to albumin in isolated coronary vessels. Am J Physiol. 1993; 265:H543-552.
2. Huxley VH, Curry FE, Adamson RH. Quantitative fluorescence microscopy on single capillaries: alpha-lactalbumin transport. Am J Physiol. 1987; 252:H188-197.
3. Schelling ME, Meininger CJ, Hawker JR Jr, Granger HJ. Venular endothelial cells from bovine heart. Am J Physiol. 1988; 254:H1211-H1217.
4. Haynes TE, Meininger CJ, Yuan Y, Granger HJ. Culture and purification of endothelial cells from isolate porcine coronary venules. FASEB. 1995; 9:A617 (abstract).
5. Gonindard C, Bergonzi C, Denier C, Sergheraert C, Klaebe A, Chavant L, Hollande E. Synhetic hispidin, a PKC inhibitro, is more cytotoxic toward cancer cells than normal cells in vitro. Cell Biol. Toxicol. 1997; 13:141-153.
6. Kashiwada Y, Huang L, Ballas LM, Jiang JB, Janzen WP, Lee KH. New hexahydroxybiphenyl derivatives as inhibitors of protein kinase C. J. Med. Chem. 1994; 37:195-200