Perioperative pulmonary hypertension is a challenging clinical problem in cardiac surgery patients. Hypoxia is a potent stimulus for pulmonary vasoconstriction (1, 2), which may be an adaptive mechanism to match lung perfusion with ventilation. Sustained hypoxic pulmonary vasoconstriction (HPV) may have detrimental effects, such as vascular remodeling, activation of proinflammatory pathways, and eventual right-sided heart failure. Hypoxic injury may also activate proinflammatory signaling pathways, leading to production of inflammatory mediators such as tumor necrosis factor (TNF) α. It is now known that TNF-α can be produced by local tissues and can exert deleterious effects on these tissues in an autocrine fashion (3). This has been demonstrated in myocardium after various forms of acute injury (4), and we have correlated increased TNF-α and interleukin (IL) 1β expression with pulmonary artery (PA) dysfunction after acute hypoxia (1).
Although the exact mechanism of HPV remains unknown, there is accumulating evidence that protein kinases such as protein kinase C (PKC) play a role (5, 6). Protein kinase C is a regulatory enzyme activated by numerous effectors, growth factors, hormones, and neurotransmitters (7). Protein kinase C isoforms are activated by stimuli such as ischemia/reperfusion (8) and endotoxemia (9). After diacylglycerol release from the cell membrane by phospholipases, PKC is activated and exerts its effects by phosphorylating proteins at serine and threonine residues. The signaling pathways involved in generating an acute inflammatory response involve mediators such as PKC. Furthermore, PKC is known to mediate TNF-α production in macrophages (9), but its role in tissue cytokine production during hypoxia is less clear.
The PKC family can be further subdivided into three groups: classical PKC (cPKC; α, β, γ), novel PKC (δ, ε, η, θ, μ), and atypical PKC (ζ, ι, λ). Different isoforms may have tissue-specific and condition-specific effects (10). We have previously shown that HPV is mediated by PKC. However, it is unknown whether these effects are isoform-specific. Because the cPKC enzymes are calcium-dependent and HPV is also calcium-dependent, we hypothesized that cPKC would mediate HPV and hypoxia-induced proinflammatory cytokine expression.
MATERIALS AND METHODS
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 85-23, Revised 1985). All animal protocols are approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine. Male Sprague Dawley rats (Harlan, Indianapolis, Ind) weighing 250 to 350 g were allowed ad libitum access to food and water up to the time of experimentation.
Isolated PA ring preparation
Rats were anesthetized with pentobarbital (150 mg/kg i.p.). Median sternotomy was performed, and the heart and lungs were removed en bloc and placed in modified Krebs-Henseleit (KH) solution at 4°C. Under a dissecting microscope, extralobar PA branches were dissected and cleared of surrounding tissue. Right and left main branch PA were cut into 2- to 3-mm wide rings (4 per animal) and suspended on steel hooks connected to force transducers (ADInstruments, Colorado Springs, Colo) for measurement of isometric force displacement. Care was taken during this process to minimize endothelial injury by avoiding contact with the luminal surface of the arteries. Pulmonary artery rings were immersed in individual water-jacketed organ chambers containing modified KH solution bubbled with 95% O2/5% CO2 at 37°C. Krebs-Henseleit solution is a physiologic balanced salt solution containing the following (in millimolar): NaCl, 127; KCl, 4.7; NaHCO3, 17; MgSO4, 1.17; KH2PO4, 1.18; CaCl2, 2.5; and D-glucose, 5.5. Force displacement was recorded using a PowerLab (ADInstruments) eight-channel data recorder on an Apple iMac PowerPC G4 Computer (Apple Computer Co, Cupertino, Calif).
Experimental protocol and groups
Before starting experimental protocols, PA rings were stretched to a predetermined (11) optimal passive tension of 750 mg and allowed to equilibrate for 60 min, during which time KH solution was changed every 15 min. Viability of each PA ring was then checked by measuring contractile response to 80 mmol/L KCl. This dosage was determined to produce maximal contractile response to KCl in previous experiments (11). After washout of KCl, endothelial integrity of each PA ring was assessed with relaxation to acetylcholine (1 μmol/L) after phenylephrine (1 μmol/L) precontraction. These concentrations were derived from preliminary experiments to produce optimal contraction and relaxation. Rings demonstrating less than 50% vasorelaxation to acetylcholine were discarded. After washout of acetylcholine, PA rings were precontracted with phenylephrine, and hypoxia was induced by changing the gas to 95% N2/5% CO2. Hypoxia was induced in PA rings (n = 6 per group) pretreated with the nonspecific PKC inhibitor bisindolylmaleimide (BIM, 1 μmol/L), the cPKC inhibitor Gö 6976 (12-[2-cyanoethyl]-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]-carbazole; 1 μmol/L), or vehicle (0.001% dimethyl sulfoxide [DMSO]) 20 min before the onset of hypoxia. Experiments were terminated after 60 min of hypoxia, and the rings were immediately snap frozen in liquid nitrogen for subsequent messenger RNA (mRNA) analysis.
Reverse transcriptase-polymerase chain reaction
Semiquantitative reverse transcriptase-polymerase chain reaction (PCR) was used to assess TNF-α and IL-1β gene expression in PA rings (n = 4 - 6 per group). After tissue homogenization, total RNA was extracted from each PA segment using RNA STAT-60 (Tel-Test, Inc, Friendswood, Tex). Total RNA (0.1 μg) was subjected to complementary DNA synthesis using a cloned avian myeloblastosis virus first-strand complementary DNA synthesis kit (Maxim Biotech Inc, South San Francisco, Calif). Complementary DNA from each sample was used for PCR of cytokines using message screen rat PCR kits (Maxim Biotech Inc). The PCR products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide. Gels were digitally photographed under ultraviolet illumination with a FotoAnalyst Luminary cooled camera electronic documentation system (Fotodyne Inc, Hartland, Wis). Gel densitometries were quantified using ImageJ software (National Institutes of Health).
Chemicals and reagents
All chemical reagents were obtained from Sigma Chemical Co (St. Louis, Mo), unless otherwise specified. All reagents were dissolved in deionized distilled water, unless otherwise specified. The cPKC inhibitor Gö 6976 was obtained from Calbiochem (San Diego, Calif) and dissolved in DMSO to make stock solutions (10 mmol/L), which were then serially diluted in deionized distilled water. All drug concentrations were expressed as final molar concentration in the organ bath. Final pH of all solutions was 7.35 to 7.45. All reagents were dissolved in deionized distilled water, unless otherwise specified.
Vasodilation was expressed as the percentage difference from the force caused by phenylephrine precontraction. Force displacement during hypoxia was expressed as percentage change from the amount of phenylephrine precontraction. All reported values were mean ± SEM. Experimental groups were compared using 2-way analysis of variance with post hoc Bonferroni test or unpaired Student t test (Prism 4, Graphpad Software, San Diego, Calif). A P value of less than 0.05 was considered statistically significant.
Hypoxic pulmonary vasoconstriction
Twenty minutes before the onset of hypoxia, the effect of PKC inhibition on acute HPV was examined by pretreating PA rings with vehicle (DMSO), the nonspecific PKC inhibitor BIM, or the cPKC inhibitor Gö 6976. Bubbling the organ baths with 95% N2/5% CO2 produced a Po2 of 30 to 35 mmHg. Acute hypoxia resulted in a biphasic PA contraction-an initial transient contraction followed by a delayed sustained contraction. There was no difference between the groups in the magnitude of the early transient contraction (data not shown). Compared with vehicle-treated PA, BIM (1 μmol/L) significantly attenuated delayed hypoxic contraction (44.59 ± 10.52% vs. 87.06 ± 10.91%) (Fig. 1). On the other hand, Gö 6976 (1μmol/L) had no effect on delayed contraction (82.61 ± 10.51% vs. 87.06 ± 10.91%) (Fig. 2A). To exclude the possibility of submaximal dosing, a 10-fold concentration of Gö 6976 (10 μmol/L) was also tested (Fig. 2B), and this dosage also failed to inhibit delayed hypoxic contraction (81.18 ± 11.02% vs. 87.06 ± 10.91%).
TNF-α and IL-1β expression by PA tissue during hypoxia
Pulmonary artery rings exposed to hypoxia were homogenized and subjected to reverse transcriptase-PCR for measurement of TNF-α and IL-1β mRNA expression. For comparison, control PA rings were incubated in the organ baths under the same conditions and maintained on normoxic gas for an equivalent period. Hypoxia resulted in increased TNF-α and IL-1β expression compared with normoxic controls (Figs. 3 and 4). Both BIM and Gö 6976 significantly inhibited the hypoxic upregulation of TNF-α. Although BIM and Gö 6976 pretreatments resulted in a trend toward decreased IL-1β mRNA during hypoxia, this did not reach statistical significance.
We demonstrated that nonspecific PKC inhibition attenuated both HPV and PA cytokine expression, whereas cPKC inhibition downregulated hypoxia-induced PA TNF-α expression but had no effect on HPV. These results suggest that HPV and hypoxia-induced PA cytokine expression are independent processes.
Hypoxic pulmonary vasoconstriction has been extensively studied, yet the exact mechanisms are still unclear. We have previously shown that PKC mediates both HPV and inflammatory cytokine expression from the PA (1). In the previous study, the nonspecific PKC inhibitor chelerythrine inhibited both HPV and hypoxia-induced PA cytokine expression. These results were reproduced in the current study using the nonspecific PKC inhibitor BIM. There is accumulating evidence that differentiates the unique roles of the various PKC isoforms (10, 12). Different stimuli within an organ or tissue may activate different isoforms. For example, hypoxia in the heart resulted in translocation of PKC α, β, γ, and ζ, whereas oxidative stress caused α, β, and ζ translocation (12). Thus, we attempted to further delineate the specific PKC isozymes involved in hypoxic pulmonary injury.
Protein kinase C activation causes vascular smooth muscle contraction by increasing the influx of calcium, and hypoxic contraction via PKC activation may likewise involve calcium influx. An increase in cytosolic calcium concentration seems to be a key event in HPV. Calcium accumulation occurs by release from intracellular stores such as the sarcoplasmic reticulum or influx through voltage-dependent channels. Investigators have shown that blocking calcium channels (13-15), depletion of intracellular calcium stores (15-18), and removing extracellular calcium (13, 19) inhibit hypoxic contraction, whereas calcium agonists potentiate HPV (20). Because HPV seems to be a calcium-dependent process (14, 17, 21, 22), we hypothesized that the cPKC isoforms (which are calcium-dependent) would be largely responsible for the effects of acute hypoxia on the PA. However, pretreatment with the specific cPKC inhibitor Gö 6976 (1μmol/L) had no effect on HPV. The question then became whether this dose of Gö 6976 was adequate to cause an inhibitory effect. A 10-fold higher dose (10 μmol/L) also had no significant effect on HPV. This suggests that the non-cPKC enzymes are involved in HPV, and there is evidence that the novel PKC enzymes may mediate HPV. Indeed, Littler et al. (23) demonstrated that loss of the PKC ε isoform resulted in attenuated HPV.
We have previously shown that hypoxia upregulated the expression of TNF-α and IL-1β from PA tissue, and this response was inhibited by nonspecific PKC inhibition (1). The correlation between HPV and hypoxia-induced inflammatory cytokine expression from the PA suggested that these processes were related and mediated by PKC activation. In fact, previous studies have shown that inflammatory cytokines potentiate HPV (24). This led us to hypothesize that inhibition of HPV would also result in attenuation of hypoxia-induced PA TNF-α and IL-1β expression, and vice versa. This was not the case in the current study because cPKC inhibition with Gö 6976 attenuated TNF-α expression but had no significant effect on HPV. Thus, HPV and hypoxia-induced PA cytokine expression seem to be independent processes. Although a significant difference in TNF-α mRNA levels was found in this study, we do not know whether protein expression was similarly affected. It is possible that TNF-α gene and protein levels do not change proportionally in response to hypoxia and PKC inhibition. Thus, we can only conclude that TNF-α gene expression was affected.
In summary, HPV is dependent on PKC activation, but the isoform involved is likely a nonclassical subtype. Hypoxia-induced upregulation of proinflammatory cytokine expression from the PA is also dependent on PKC activation, and this does seem to involve cPKC isoforms. Contrary to previous studies, HPV and PA cytokine upregulation can occur independently, which suggests that the two processes are not mechanistically linked. Further differentiation of PKC isoform specificity may help identify potential therapeutic targets in the treatment of hypoxic pulmonary injury.
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Hypoxia; pulmonary hypertension; inflammation; signal transduction