1Cardiovascular Pulmonary Research Laboratory and 2Department of Pathology, University of Colorado Health Sciences Center; and 3Denver Veterans Administration Medical Center, Denver, Colorado; and 4Ernest Gallo Clinic and Research Center, Department of Neurology, University of California San Francisco, Emeryville, California
Submitted 22 December 2004 ; accepted in final form 25 July 2005
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ABSTRACT |
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mouse; knockout; pulmonary hypertension; endothelial; inducible and neuronal nitric oxide synthase; vascular remodeling; protein kinase C epsilon
The right ventricle (RV) responds to PHTN with an adaptive hypertrophic response, the magnitude of which typically parallels the pressure elevation in the pulmonary circulation (33). In a few instances, uncoupling of the extent of PHTN and cardiac hypertrophy has been observed (41, 43). These reports suggest that mechanisms regulating adaptive responses of PA smooth muscle cells (SMC) and RV myocytes (including hypertrophy/hyperplasia) may be different.
Protein kinase C (PKC) is an important signaling intermediate in many of the vascular cell responses (like contraction and growth) thought to be important in chronic hypoxic PHTN. The PKC family includes at least 10 related intracellular kinases (,
I,
II,
,
,
,
,
,
/
,
) that are classified into three groups and share varying degrees of homology (5, 30). Another related but distinct subfamily of protein kinases (PKD/PKC-µ, PKD2, and PKD3/PKC-
) has also recently been characterized (38). The role of individual PKC isozymes in the regulation of susceptibility to chronic PHTN has remained unclear. The PKC-
isozyme is likely important since it regulates myocardial responses to ischemia (13). We have also recently found that PKC-
depletion protects against acute HPV by allowing increased KV channel expression (27). This response could not be attributed to a general loss in vasoreactivity or derangements in nitric oxide synthase (NOS) isoforms. These observations suggest that loss of PKC-
could protect the pulmonary circulation from PHTN induced by chronic Hx. However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response (8, 22). Adaptation of the lung circulation to chronic Hx is a more complex process than acute HPV and likely involves additional pathways and intermediates.
Decreased expression/activity of NOS contributes to many models of vascular injury and dysfunction (26). Although we did not find that basal differences in NOS could account for the blunted HPV found in PKC-/ mice exposed to acute Hx (28), abnormalities in NOS expression/activity could still be important in adaptive responses to chronic Hx in this model (23). All three NOS isoforms [endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)] are expressed in rodent lung (11, 24). Lung eNOS and iNOS expression increase in response to chronic Hx in mice and rats (24). NO, derived from eNOS, and to a lesser extent iNOS, is thought to be the major mediator of vasodilation in the murine pulmonary circulation (11).
We used a knockout mouse approach to test the hypothesis that the absence of PKC- would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic Hx. Gray and colleagues (13) recently completed an extensive cardiac assessment of the PKC-
+/+ and PKC-
/ mice used in the current study. Their analysis included direct inspection, echocardiography, and left heart catheterization. They found no differences in cardiac structure or function at baseline or after acute ischemia reperfusion. These findings suggested that right ventricular systolic pressure (RVSP) measurements made here would provide a useful, albeit indirect, index of PA pressure. Interestingly, we found that chronically hypoxic PKC-
/ mice actually have increased PHTN, RV hypertrophy (RVH), and polycythemia without further pulmonary vascular remodeling compared with PKC-
+/+ mice. PKC-
+/ mice had an intermediate RVSP and RVH response. However, reactive polycythemia was no different in these mice than PKC-
+/+ controls. Thus the remodeling in the PKC-
/ and PKC-
+/ pulmonary circulation was less than expected, considering the pressure elevation and extent of RVH. An acute vasodilator response to inhaled NO was then measured in additional chronically hypoxic PKC-
+/+ and PKC-
/ mice to determine the contribution of pulmonary vasoconstriction vs. structural remodeling to the augmented RVSP response seen under chronically hypoxic conditions. An enhanced vasodilator response to inhaled NO in the more hypertensive PKC-
/ pulmonary circulation would strongly suggest that an increase in pulmonary vascular resistance was responsible for the elevated RVSP detected in chronically hypoxic PKC-
/ mice. Because differences were observed, a comparison of NOS expression and activity was undertaken. Collectively, the findings from these studies indicate roles for PKC-
in pulmonary vascular reactivity, remodeling, and induction of NOS isoforms during chronic Hx.
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MATERIALS AND METHODS |
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Animals.
Adult female C57BL6 x SV129 PKC- wild-type (PKC-
+/+), heterozygous null (PKC-
+/), and homozygous null (PKC-
/) mice were obtained as described (18, 21). F1 hybrid mice were generated at the Gallo Research Center (Emeryville, CA; sea level altitude) by crossing PKC-
+/ SV129 females with wild-type C57BL6 males. Resulting F1 generation PKC-
+/ mice were then bred to generate PKC-
+/+, PKC-
+/, and PKC-
/ F2 offspring that were then sent to Denver (altitude 5,280 ft) at 1113 wk of age for experiments. These mice were allowed to acclimate to this altitude for 5 wk before study. Animal protocols were approved by the University of Colorado Health Sciences Center Institutional Animal Care and Use Committee.
PKC- expression in mouse whole lung homogenates.
To examine differences in PKC-
expression in PKC-
+/+, PKC-
+/, and PKC-
/ mice, quick-frozen lung tissue was weighed and homogenized on ice (PowerGen 700, Pittsburgh, PA) in 10 volumes of homogenization buffer [10 mM Tris pH 7.4 containing 0.15 M NaCl, 40 mM NaF, 100 µM Na orthovanadate, 1.0% Triton X-100, 1.0% Nonidet P-40, and a 1.0% protease inhibitor cocktail including 1.04 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 0.8 µM aprotinin, 21 µM leupeptin, 15 µM pepstatin A, 36 µM bestatin, and 14 µM E-64]. The homogenates were centrifuged at 1,500 g for 20 min at 4°C (Savant µSpeedFuge, Holbrook, NY), and supernatant was retained. Whole lung homogenate (50 µg) was loaded per lane of an 8% reduced SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and processed as previously described (27). Representative mouse lung lysates subjected to Western blotting are shown in Fig. 1. The relative amount of PKC-
protein in lungs from PKC-
+/ mice was found to be 52 ± 7.5% of PKC-
+/+ levels [n = 7 mice; current data pooled with that previously reported (27)].
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Blood and tissue harvesting.
Blood samples were routinely taken from the RV via percutaneous stick at the end of the hemodynamic measurements via a separate syringe and 25-gauge needle. The hematocrit was measured with a capillary tube and standard techniques. Collected blood samples were allowed to coagulate for 1 h, and serum was collected after centrifugation. After baseline hemodynamic measurements, the chest was opened, and 100 units of heparin were injected into the RV. Lungs were isolated, inflated, and fixed as previously described with modifications (11). The pulmonary circulation was gently perfused at a pressure of 40 cmH2O with PBS to remove blood. The left bronchus was ligated, and the left lung was removed and quick-frozen in liquid nitrogen. A slit was made in the trachea, and a 16-gauge tubing adapter was inserted and tied in place. A syringe filled with 1% prewarmed agarose (GIBCO, Grand Island, NY) and 1% paraformaldehyde in PBS was attached to the adapter. An inline pressure transducer (Statham) with Biopac recorder was used to monitor inflation pressure in the airway in selected studies (n = 5 mice). The right lung was routinely inflated over
70 s to a highly reproducible visual endpoint (tip of the right lower lobe was completely inflated) and an airway pressure of 30.5 ± 3.9 cmH2O. In additional control studies, local tissue pressure in the peripheral lung before or after inflation was measured in separate mouse lungs by pleural puncture with a 25-gauge needle attached to the same pressure transducer. Baseline pressure was 2.1 cmH2O and consistently increased to 9.3 ± 0.5 cmH2O with the technique described (n = 4 mice). A blinded lung pathologist (C. D. Cool) examined sections from the six experimental groups and judged the quality and consistency of inflation and fixation to be excellent across all groups. The trachea was tied off, and the lung was removed and placed in cold 2% paraformaldehyde/PBS for 2 h, cut, and placed in cassettes in 10% buffered formalin for up to 24 h before being embedded in paraffin. In selected experiments the heart was removed and fixed in 10% buffered formalin (VWR, West Chester, PA) before being embedded in paraffin.
Measurement of RVH. Immediately following removal of inflated lungs, hearts were routinely removed and weighed. The atria were trimmed away and the right ventricular free wall (RV) was dissected from the left ventricle (LV) and septum (S). Both ventricular sections were weighed wet and then reweighed after being air-dried for 7 days. A blinded pathologist (C. D. Cool) reviewed cross sections of hearts from the six experimental groups and found the quality of fixation to be excellent.
Tissue staining.
Sections (4 µm) of embedded mouse lung and heart tissues were deparaffinized at 60°C for 45 min and then rehydrated. Tissues were placed in 0.5% hydrogen peroxide (Fisher Scientific, Fairlawn, NJ) for 5 min, washed, and blocked in PBS containing 1% horse serum, 0.6% Triton 100 and avidin (Vector Lab, Burlingame, CA) for 2 h. Hematoxylin and eosin (H&E) staining was performed on lung and heart sections from PKC-+/+, PKC-
+/, and PKC-
/ mice to screen for structural differences.
A double stain was used to quantify the subtle pulmonary vascular structural changes observed in murine chronic hypoxic PHTN. An endothelial cell marker, factor VIII, was used to detect pulmonary vessels. -Smooth muscle (
-SM) actin, a marker for SMC and myofibroblasts, was used to detect medial and adventitial thickening of proximal vessels and muscularization of distal vessels. Tissue was incubated with a 1:1,000 dilution of anti-factor VIII (Dako A0082; Dako, Glostrup, Denmark) and biotin (Vector Lab) overnight (ON) at 4°C. Control slides received no primary antibody. To detect bound primary antibody, we incubated slides at 1:400 for 1 h in biotinylated anti-rabbit IgG (Vector Lab). Slides were washed in PBS and incubated in Elite ABC solution (Vector Lab) for 1 h. After another wash, slides were developed with diaminobenzidine substrate (Vector Lab). Tissues were blocked with PBS/horse serum/Triton X-100 for 30 min and then incubated in anti-
-SM actin antibody (Immunon 079a; Shandon, Pittsburgh, PA) 1:30 for 12 min. Slides were next incubated with biotinylated anti-mouse antibody 1:200 for 1 h and, finally, incubated for 30 min in alkaline phosphatase standard. Slides were rinsed in PBS and developed in alkaline phosphatase substrate. Slides were counterstained with 25% Gills hematoxylin #1 (Fisher Scientific) and coverslipped with Immumount (Shandon).
Morphometric analysis.
Using Stereo Investigator software (MicroBrightField, Colchester, VT), we measured medial wall thickness of 50125 µm proximal vessels by calculating the difference between the outer edge of the media [or the external elastic lamina (EEL)] and the inner edge of the media (or the internal elastic lamina) at four points around the vessel circumference and determining an average of these values. The percent medial thickness for these vessels was calculated as the average medial wall thickness divided by the EEL diameter and presented as a percentage. Distal pulmonary vessels (between 10 and 50 µm outside diameter) were assessed by a blinded observer for degrees of circumferential -SM actin-positive staining indicative of muscularization. Vessels were scored as none (0% of circumference actin positive, NM), partially (174%, PM), and fully (75100%, FM) muscularized. Vessels smaller than 10 µm were considered capillaries and excluded from further consideration. For purposes of data presentation PM and FM vessels were combined together and reported as total muscularized vessels. We calculated the value for percent muscularized vessels by taking the number of PM and FM vessels and dividing by the total number of factor VIII-positive distal vessels counted. We calculated vessel density by taking the number of factor VIII-positive distal vessels counted and dividing by the area of inflated lung examined.
NO inhalation studies.
NO, an important regulator of vascular tone, was selected for the vasodilator challenge. These nonsurvival studies required stand alone Nx and Hx as well as NO and +NO treatment groups. The inhaled NO experiments were performed as previously described (9). In brief, a hood flushed with a gas mixture of 21% O2 plus 25 ppm NO was placed over the head of each mouse for 5 min, and closed-chest hemodynamic measurements were performed as described in Hypoxic exposure and hemodynamic measurement. The NO concentration was monitored by continuous gas analysis (Pulmonox II; Pulmonox Medical, Tofield, Alberta, Canada). Because of the effectiveness of inhaled NO in the PKC-/ mice, no other agents were tested.
NOS isozyme expression in mouse whole lung homogenates and tissue sections.
Quick-frozen lung tissue was weighed and homogenized on ice and processed as described for measurement of PKC- expression. Whole lung homogenate (5080 µg) was loaded per lane of an 8% reduced SDS-polyacrylamide gel, and positive controls for iNOS [lipopolysaccharide (LPS)/PMA-activated RAW 264.7 murine macrophage cell lysates; BD Transduction Laboratories, San Diego, CA] and nNOS (mouse brain extract; Santa Cruz Biotechnology, Santa Cruz, CA) were used and transferred to a nitrocellulose membrane as previously described (27). Molecular weight protein markers (Bio-Rad, Hercules, CA) were also loaded into the first lane of each gel.
Anti-eNOS and phospho-specific (p)-eNOS antibodies were purchased from BD Transduction Laboratories, and anti-nNOS antibody from Zymed (South San Francisco, CA). Using LPS/PMA-activated RAW 264.7 murine macrophage cell lysates as a positive control, we compared anti-iNOS antibodies from BD Transduction Laboratories (polyclonal and monoclonal antibodies), Affinity Bio Reagents, Biomol, and Santa Cruz Biotechnology under optimal conditions per manufacturer instructions for sensitivity and specificity by Western blot. The polyclonal iNOS antibody from BD Transduction Laboratories was found to be the most sensitive and specific and was used for all experiments.
After a 1-h incubation at room temperature (RT, 37°C) in 2% BSA/Tris-buffered saline (TBS)/0.05% Tween 20 [for eNOS and (Ser-1177 and Thr-495) p-eNOS detection] or ON incubation at 4°C in 5% dry milk/TBS/0.05% Tween 20 (for nNOS and iNOS detection) to block nonspecific binding, the nitrocellulose was probed with eNOS (1:1,000), p-eNOS (1:1,000), nNOS (1:1,000), or iNOS (1:1,000) antibodies ON at 4°C. Blots were incubated for 1 h at RT with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG at a dilution of 1:5,000 in either 2% BSA/TBS/0.05% Tween 20 (eNOS and p-eNOS) or 5% milk/TBS/0.05% Tween 20 (nNOS and iNOS). The nitrocellulose was washed again and developed as previously described (27).
iNOS staining was performed on lung sections from PKC-+/+ and PKC-
/ mice to screen for any differences in expression since only the positive control was detected by Western blot. Antigen retrieval was performed by the heat-induced epitope retrieval method, and antigen was blocked in avidin, biotin, and Powerblock (BioGenex, San Ramon, CA) each for 15 min. Tissue was routinely incubated with a 1:500 dilution of anti-iNOS antibody (BD Bioscience, San Diego, CA) and 1.0% horse serum in TBS ON at 4°C. In selected experiments, serial dilutions of primary antibody were applied from 1:250 to 1:3,000. The negative control slides received no primary antibody. To detect bound primary antibody, slides were washed and then incubated for 12 h in goat anti-rabbit IgG (1:400). Slides were washed and incubated in Elite ABC solution (Vector Lab) for 1 h. After further washing with TBS (2x 5 min), slides were developed, counterstained, and mounted as described in Tissue staining.
NO metabolite measurements. Serum samples obtained via transthoracic cardiac stick following hemodynamic measurements were thawed and centrifuged at 15,000 g for 20 min when ready to measure NO metabolites. Serum NO metabolites including NO, NO2, NO3, peroxynitrate, and nitrosothiols were measured in triplicate with a NO chemiluminescence analyzer (Ionics Instrument Business Group, Boulder, CO) as previously described (10, 35).
Data analysis.
All data were expressed as means ± SE. For all studies reported here, n represents the number of individual mice on which measurements were made. In some instances the mean of multiple measurements was used to generate the value for each individual mouse. For hemodynamic studies, exclusion criteria included equipment problems (i.e., transducer failure) or the presence of relative bradycardia [heart rate <250 beats/min (bpm)]. Proximal vessel wall thickness measurements were made on at least 10 vessels in the 50- to 125-µm size range per mouse. For measurement of extent of distal muscularization and vessel density at least 100 vessels were analyzed in the 10- to 50-µm size range per mouse. Mechanistic studies were limited to PKC-+/+ and PKC-
/ mice. Statistical comparisons were completed using the SuperANOVA software program (Abacus Concepts, Berkeley, CA) between genotype (PKC-
+/+, PKC-
+/, and PKC-
/) and treatment (Nx and Hx) groups. Comparisons were performed by two-way analysis of variance (ANOVA) followed by the Scheffés multiple-comparison tests for individual comparisons within and between groups of data points. Data were considered significantly different if P < 0.05.
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RESULTS |
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Effect of chronic Hx on pulmonary vascular structure in PKC-+/+, PKC-
+/, and PKC-
/ mice.
To determine the effect of reduced PKC-
on pulmonary vascular structure at baseline and in response to chronic Hx, morphometric analysis of both proximal (50125 µm) and distal (1050 µm) vessels was performed on PKC-
+/+, PKC-
+/, and PKC-
/ mouse lung sections following double staining for endothelial (factor VIII) and smooth muscle (
-SM actin) cells. Measurements of medial wall thickness of proximal vessels in PKC-
+/+, PKC-
+/, and PKC-
/ mouse lungs were not dependent on vessel density calculations (Table 1). At baseline no differences in wall thickness across the three genotypes were detected. A subtle increase in the percent medial wall thickness was found in PKC-
+/+, PKC-
+/, and PKC-
/ mice exposed to chronic Hx. The signal was of similar magnitude across all three genotypes and, when pooled, was significantly greater than the aggregate normoxic response. Thus despite the increased RVSP and RVH response, PKC-
/ and PKC-
+/ mice did not have increased pulmonary vascular remodeling of their proximal vessels compared with PKC-
+/+ mice. In a separate review, a blinded pulmonary vascular pathologist detected only a subtle difference in proximal vessels between Nx- and Hx-treated groups upon review of H&E-stained slides (C. D. Cool). No further differences between genotypes within these treatment groups were found. PKC-
/ and PKC-
+/ mice also did not have increased vascular remodeling in their distal vessels (i.e., muscularization) compared with PKC-
+/+ mice following chronic Hx (Fig. 5). Measurements of distal muscularization (PM + FM divided by total vessels x 100) were also not dependent on vessel density calculations. Vessel density (total 10- to 50-µm-diameter vessels divided by unit area of lung examined) was similar in PKC-
+/+, PKC-
+/, and PKC-
/ mouse lungs at baseline (Nx: PKC-
+/+ 13.3 ± 1.3, PKC-
+/ 12.6 ± 2.1, PKC-
/ 16.3 ± 2.4 vessels/µm2; P = not significant; n = 69 mice/group). After chronic Hx, the calculated density of 10- to 50-µm-diameter vessels in PKC-
/ lungs was lower than was found in normoxic PKC-
/ lungs (Hx: PKC-
+/+ 14.0 ± 2.7, PKC-
+/ 16.9 ± 1.7, PKC-
/ *10.4 ± 0.9 vessels/µm2; *P < 0.05 vs. Nx PKC-
/; n = 69 mice/group).
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DISCUSSION |
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The reversibility with NO suggested that inadequate baseline and/or Hx-induced expression/activity of one or more of the NOS isoforms might be contributing to the divergent responses seen here. Decreased NOS expression/activity has been found in models of systemic vascular injury (12, 24). Fagan et al. (11) have shown that NO derived from eNOS and to a lesser extent iNOS is the major mediator of vasodilation in the murine pulmonary circulation. Ping et al. (32) have shown that the eNOS and iNOS isoforms are physically associated with PKC- in the heart, suggesting a novel role in the modulation of these two NOS isoforms in myocytes. We found that baseline eNOS and iNOS protein expression by Western blotting and immunostaining, respectively, was similar in PKC-
+/+ vs. PKC-
/ lungs. We also found a trend toward lower nNOS expression in PKC-
/ vs. PKC-
+/+ lungs at baseline. However, this isoform of NOS does not have a known role in pulmonary vascular biology and probably is more important in airway function. Our findings in PKC-
+/+ mice following chronic Hx are consistent with previous reports that expression of eNOS increases with chronic hypoxic PHTN in rodents (24). After 35 days of Hx, eNOS expression was induced to a greater extent in PKC-
+/+ than PKC-
/ mice. We also found, by immunostaining, a trend that iNOS expression was also increased more in PKC-
+/+ than PKC-
/ lungs.
The results support a role for PKC- in the regulation of Hx-induced expression of eNOS rather than in the direct regulation of enzymatic activity by phosphorylation of the enzyme. PKC-
+/+ and PKC-
/ lungs showed similar p-eNOS expression of the Thr-495 residue under normoxic and chronic hypoxic conditions, suggesting eNOS activity was not decreased relative to expression by this mechanism. These findings imply that PKC-
is important in the regulation of Hx-induced expression of eNOS rather than enzymatic activity. Li et al. (25) showed that various PKC inhibitors prevent phorbol ester-induced enhancement of eNOS expression; furthermore, this enhancement seemed to be a transcriptional event. This observation suggested PKC is important in the induction of eNOS expression but did not implicate a specific isozyme, nor was it necessarily relevant to hypoxic responses.
The regulation of eNOS activity is complex and involves several kinases including PKC, PKA, Akt, and AMP-activated protein kinase as well as two phosphatases including PP1 and PP2A (29). PKC signaling has been implicated in eNOS protein phosphorylation at Thr-495 as well as promoting dephosphorylation of the protein at Ser-1177. These events result in decreased eNOS activity in bovine aortic as well as human umbilical vein endothelial cells by inhibiting the interaction between eNOS and calmodulin (28, 29). However, the specific PKC isozyme involved in this regulation of eNOS activity has not been determined, as only general PKC activation by phorbol 12,13-dibutyrate treatment has been observed. We thought it was possible PKC- was involved here; however, our results do not support this.
NOx measurements are similar to the results of the p-eNOS (Ser-1177 and Thr-495 residues) blotting, which suggests that eNOS protein expression (but not activity per unit protein) was increased following Hx. The NOx measurements in PKC-+/+ mice are also consistent with similar studies that showed that rats breathing 10% O2 chronically did not have an increase in NO plasma metabolite levels, suggesting that Hx inhibited NO production despite increases in eNOS isoform expression (35). Limiting factors here are the sampling site (final RV stick after initial hemodynamic measurements with potential for mixing with arterial blood from the LV), the single 35-day time point, and the recent studies showing NOx measurements, regardless of site, may not be that informative (17). Although serum NOx concentrations have been shown to correlate with erythrocyte NOx concentrations, a recent study suggests that steady-state NOx concentrations reflect renal excretion of NOx metabolites rather than NOx formation by endothelial cells in vivo (17).
The immunostaining data also support a role for PKC- in the regulation of Hx-induced expression of iNOS. Interestingly, macrophages from PKC-
/ mice have an attenuated response to LPS and interferon with decreased generation of NO (1). Macrophages from these PKC-
/ mice fail to induce iNOS expression, suggesting that PKC-
is important in cytokine-induced expression of iNOS. Our observation of a blunted increase in iNOS expression in PKC-
/ mice exposed to chronic Hx is also consistent with a previous report in RAW 264.7 murine macrophages suggesting that PKC-
is sufficient to induce iNOS synthesis via a mechanism that involves activation of response elements in the iNOS promoter region (7).
Reactive polycythemia is probably also contributing modestly to the increased PA pressure detected in PKC-/ mice. Polycythemia has been shown to increase blood viscosity and pulmonary vascular resistance (16). Reactive polycythemia has only a small effect on PA pressure and takes several weeks to detect. Walker et al. (37) showed that administration of human recombinant erythropoietin (rEpo) for 2 wk to rats to increase hematocrit from 45 to 66% made no difference in mean PA pressure. At 4 mo of age, mice that chronically overexpress Epo (10-fold over control) and have severely elevated hematocrit (86 vs. 45 for controls) have only modestly elevated PA pressures (22 vs. 15 for controls) (15). Finally, Petit et al. (31) have shown that potentiation of hematocrit in chronically hypoxic rats from 63 to 72% with rEPO (a scenario similar in some ways to what we are observing here) failed to further enhance PHTN. Polycythemia could alter eNOS expression or activity, thereby altering vascular reactivity. There are conflicting reports that polycythemia increases eNOS expression and NO production (16, 37). Walker et al. (37) showed that in rats administered rEpo, hematocrit levels were found to be increased compared with control; however, the increase in shear stress associated with this polycythemia did not increase eNOS expression or NO metabolites. The Epo-overexpression mouse with severely elevated hematocrit has increased eNOS expression (15, 16). If anything, these studies reinforce the importance of the blunted NOS response we observed.
Despite the increased RVSP and RVH response, PKC-/ and PKC-
+/ mice did not have increased pulmonary medial remodeling. Medial wall thickness of proximal vessels was subtly increased to the same extent in all three genotypes following chronic Hx. The distal vasculature also did not show increased pulmonary remodeling. Neither measurement was dependent on vessel density calculations. The extent of remodeling we observed was less than the maximal levels reported for other genetically altered mice, suggesting that a ceiling for extent of structural change had not yet been reached (19). Collectively, these findings demonstrate that the contractile and structural responses of the murine lung circulation to Hx can be uncoupled from one another. The altered vascular reactivity may be at least partly due to a decrease in PKC-
protein expression. Less medial remodeling than expected has been observed in a few other settings, for instance, when rEpo is chronically administered or overexpressed in mice (15, 16). Because apoptosis helps limit medial smooth muscle remodeling and Epo is antiapoptotic, the exact mechanism here is unclear. We speculate another possibility may be operative in our PKC-
/ mice. PKC-
has been implicated in the signaling pathway that leads to induction of the heat shock protein (hsp) 90
gene in response to stimuli (40). The hsp90
has been shown to help regulate cell growth and may protect cells from apoptosis (40). Thus a deletion of the PKC-
gene may lead to decreased activity of the hsp90
protein and induction of SMC apoptosis (limiting medial remodeling) following an extreme stress such as chronic Hx.
Chronic Hx may have caused a modest decrease in density of vessels in PKC-/ mice compared with normoxic counterparts. This could contribute to the exaggerated RVSP seen in PKC-
/ mice exposed to chronic Hx. However, the lower density would not likely contribute to the decrease in RVSP found with NO inhalation (i.e., the reversible component). PKC-
deletion could predispose vascular endothelial cells to apoptosis causing vessel rarification. Consistent with this idea is the finding that eNOS expression is lower than expected following chronic Hx in PKC-
/ mice. In other systems, eNOS is thought to be protective against apoptosis (3, 12).
We showed that the PKC-/ and, to a lesser extent, PKC-
+/ mice have increased RVH compared with PKC-
+/+ mice. Clearly, decreased PKC-
expression does not limit the adaptive responses of the RV to increased pressure. Several studies have shown that activation of PKC-
is important in protection of cardiac myocytes from ischemic/hypoxic injury (4, 13, 32). PKC-
has been shown to be necessary and sufficient to confer cardioprotection (32). Thus the role of PKC-
in adaptive responses to hypoxia may be tissue specific.
In summary, our results demonstrate that the contractile and structural responses of the murine lung circulation to chronic Hx can be uncoupled from one another and from RVH. Chronically hypoxic PKC-/ mice have a deficiency in Hx-induced eNOS expression in the lung that probably contributes to the increased PHTN detected. PKC-
is likely an important intermediate in the regulation of Hx-induced eNOS as well as iNOS expression. Loss of PKC-
provides relative protection from exaggerated pulmonary vascular, but not RV, remodeling in response to chronic Hx. These findings suggest that PKC-
is an important determinant of susceptibility to chronic hypoxic PHTN.
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GRANTS |
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ACKNOWLEDGMENTS |
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Preliminary results published in abstract form only: Western Student Medical Research Forum, Carmel, CA (J Invest Med 52: C452, 2004); American Thoracic Society, Orlando, FL (Am J Respir Crit Care Med 169: A399, 2004); and Thomas L. Petty Aspen Lung Conference on Cellular and Molecular Pathobiology of Pulmonary Hypertension, Aspen, CO (Chest, in press).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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