Departments of 1 Surgery and 2 Physiology, Wayne State University, Detroit, Michigan 48201
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ABSTRACT |
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Lipopolysaccharide (LPS)-regulated
contractility in pericytes may play an important role in mediating
pulmonary microvascular fluid hemodynamics during inflammation and
sepsis. LPS has been shown to regulate inducible nitric oxide (NO)
synthase (iNOS) in various cell types, leading to NO generation, which
is associated with vasodilatation. The purpose of this study was to
test the hypothesis that LPS can regulate relaxation in lung pericytes and to determine whether this relaxation is mediated through the iNOS
pathway. As predicted, LPS stimulated NO synthesis and reduced basal
tension by 49% (P < 0.001). However, the NO synthase
inhibitors N -nitro-L-arginine
methyl ester, aminoguanidine, and
N
-monomethyl-L-arginine did
not block the relaxation produced by LPS. In fact, aminoguanidine and
N
-monomethyl-L-arginine
potentiated the LPS response. The possibility that NO might mediate
either contraction or relaxation of the pericyte was further
investigated through the use of NO donor compounds; however, neither
sodium nitroprusside nor
S-nitroso-N-acetylpenicillamine had any significant
effect on pericyte contraction. The inhibitory effect of aminoguanidine
on LPS-stimulated NO production was confirmed. This ability of LPS to
inhibit contractility independent of iNOS was also demonstrated in lung
pericytes derived from iNOS-deficient mice. This suggests the presence
of an iNOS-independent but as yet undetermined pathway by which lung
pericyte contractility is regulated.
nitric oxide; sepsis; inducible nitric oxide synthase
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INTRODUCTION |
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ABNORMALITIES IN CAPILLARY permeability are hallmarks of inflammation and can lead to extravascular fluid sequestration. It has been proposed that mediators of edema increase permeability by altering the contractile state of either the smooth muscle cell or the pericyte within the vascular wall. Because increases in vascular permeability have been shown to exist in capillaries or venules that have no smooth muscle, pericyte contractility may be a factor in this response. This hypothesis has since been supported by several studies involving both morphological (3, 32) and microscopic (27) analyses. For example, Majno et al. (18) proposed that mediators of edema produced contractions within the cremaster microvessels, which created gaps of up to 1 µm in diameter that allowed leakage of macromolecules into the interstitium. Others (4, 9) have also observed similar findings of gap formation in the presence of histamine. Other investigators (3, 7, 10) have demonstrated very potent capillary constrictor effects by mediators of edema, resulting in increased capillary permeability. Whether the pericyte exerts its effect on microvascular permeability through regulation of perfusion pressure, intercellular gap junction formation, or both is still a matter of debate. Thus there is both in vitro and in vivo data to support the association of pericyte contraction and microvascular permeability. Therefore, investigation of mechanisms regulating pericyte contraction is of interest.
In the clinical setting, loss of capillary permeability regulation can lead to extravascular interstitial fluid accumulation in the lungs as seen in acute respiratory distress syndrome (ARDS), one possible sequela of the systemic inflammatory response syndrome (SIRS). Because pericytes could mediate these changes in capillary permeability, contractile responses of the pericyte may be a linchpin in this and other clinical pathophysiological conditions involving capillary dysfunction, such as hypertension and diabetes.
Lipopolysaccharide (LPS) is a proximal mediator in the initiation of
sepsis as well as of local inflammation and is a potent stimulus of
other inflammatory mediators, including nitric oxide (NO). NO is a key
player in the cytokine cascade, which has been shown in vivo to be
capable of producing peripheral hypotension. In vitro studies have also
demonstrated the ability of NO to relax both smooth muscle cells (22,
24) and retinal pericytes (11), which suggests that NO may play an
important role in regulating blood pressure as well as microvascular
permeability. One mechanism in which LPS stimulates NO production is by
upregulating inducible NO synthase (iNOS) in vascular tissue (37). Many
cells within the vasculature are capable of expressing iNOS (15, 19,
20, 28, 33), including endothelium, smooth muscle, and mesangial cells.
A recent study (16) has demonstrated the ability of LPS to attenuate
KCl-stimulated contractile responses of smooth muscle cells in vitro
through an iNOS-dependent mechanism. In another study (31), LPS was
also capable of attenuating KCl-stimulated contractile responses in
both mammary arteries and saphenous veins. The aim of this study was to
determine whether LPS can affect lung pericyte contractility and, if
so, whether this effect is mediated through iNOS. To determine iNOS
involvement, the ability of the NOS inhibitors aminoguanidine,
N-nitro-L-arginine methyl ester
(L-NAME), and
N
-monomethyl-L-arginine
(L-NMMA) to attenuate the LPS response was examined.
Nitrate and nitrite production was also measured as an indicator of NOS
activity. In addition, the ability of LPS to affect contractility in
iNOS-deficient mice was examined to demonstrate, nonpharmacologically,
that LPS reduces pericyte contractility independent of iNOS.
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MATERIALS AND METHODS |
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Culture and characterization of lung pericytes. Pericytes were isolated from rat lungs as previously described (8) and modified by this laboratory (30) to account for the complexity of lung tissue. Briefly, male Sprague-Dawley rats (250-274 g; Harlan, Indianapolis, IN) were killed by lethal injection of pentobarbital sodium (120 mg/kg) in accordance with the National Institutes of Health guidelines and approved by the Animal Investigation Committee of Wayne State University (Detroit, MI). Each lung was dissected out and rinsed with calcium- and magnesium-free PBS. To avoid possible smooth muscle cell contamination, only the outer 2-mm peripheral portion of the lung was removed. The tissue was minced with scissors and incubated at 37°C for 20 min in 10 ml of PBS containing 1,000 U/ml collagenase type I, 0.5% BSA, 2 U/ml pronase E, and 0.5 U/ml DNase (all purchased from Sigma, St. Louis, MO). After the initial incubation, the tissue was homogenized with two strokes of a pestle to loosen microvessel fragments and incubated for an additional 10 min. The resulting suspension was filtered through a 100-µm nylon mesh to remove large tissue fragments and washed with DMEM containing 10% fetal bovine serum (FBS) and 0.5% antibiotic-antimycotic solution (ABX) (all purchased from GIBCO BRL, Life Technologies, Gaithersburg, MD). After being washed, the cell pellet was resuspended in 10 ml of red blood cell lysis buffer (Sigma) for 10 min at 4°C, washed two more times, and resuspended in uncoated tissue culture dishes in DMEM supplemented with 10% platelet-deficient serum (PDS; Sigma) and 0.5% ABX at a cell concentration of ~106 cells/60-mm dish.
Supplementation of PDS in place of FBS allowed the preferential selection of pericytes so that by second or third passage, only pericytes remained. Once a homogeneous culture of pericytes was obtained, FBS was added to the pericyte plating medium in place of PDS to provide the growth factors necessary for proliferation of the pericytes. Only cells in passages 2 through 7 were used. Male C57BL /6 (control) and C57BL /6-Nos2 (iNOS-deficient) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and killed by lethal injection of pentobarbital sodium (120 mg/kg) in accordance with the National Institutes of Health guidelines and approved by the Animal Investigation Committee of Wayne State University. The pericytes were isolated from the mouse lungs in the same manner as described above for rats except that tissues from four mice were combined and treated as one. Only cells in passages 2 through 4 were used. The identity of the pericytes was confirmed by the presence of smooth muscle actin (29) observed by indirect immunofluorescent microscopy with monoclonal anti-Contractility assays. Three-dimensional collagen gel matrices were prepared as previously described (13) and modified (30). Collagen type I (Pancogene S, Gattefosse, Westwood, NJ) was dialyzed in 1% (vol/vol) acetic acid for 36 h and stored at 4°C as stock (3 mg/ml). To prepare the gels, stock collagen was added to plating medium (DMEM containing 10% FBS and 0.5% ABX) at a ratio of 1:3, mixed rapidly, plated into 24-well plates (0.5 ml/well), and incubated at 37°C for 15 min to allow the collagen to polymerize. Cultured pericytes were then passaged with 0.125% trypsin-0.02% EDTA (GIBCO BRL), plated on top of the collagen gels at a concentration of 7.5 × 104 cells/well in plating medium (0.5 ml/well) unless otherwise noted, and incubated for 24 h at 37°C and 5% CO2.
After 24 h, the medium was removed from the wells by inverting the wells over a paper towel in a sterile hood. For the LPS assays, the cells were rinsed and resuspended in assay medium (DMEM containing 400 µM L-arginine) with 15 µM BSA in the presence of various LPS concentrations (0-100 µg/ml) for 24 or 48 h at 37°C and 5% CO2. To test the effects of the NOS inhibitors, cells were incubated with 0-100 µg/ml LPS in the presence and absence of 200 µM aminoguanidine (RBI, Natick, MA), 200 µM L-NMMA (RBI), or 10 µM L-NAME (Sigma) for the entire incubation. All solutions were prepared just before use. For assays involving bradykinin (control agent; Sigma) and the NO donors spermine NONOate (Cayman Chemical, Ann Arbor, MI), sodium nitroprusside (SNP; Sigma), and S-nitroso-N-acetylpenicillamine (SNAP; RBI), a stock solution was prepared just before use by dilution in assay medium. Dilutions were added as 10× the final concentration and allowed to incubate for 10 min before the gels were detached from the sides of the well. On the day of assay, the gels were detached from the sides of their wells and photographs were taken at 10-min intervals, up to 60 min, with the aid of a macro lens. Collagen surface area was measured with Digitizer software, and contraction is expressed as percentage of the initial surface area. Results are expressed as percent change in contraction from the control value (no LPS). For measurement of NO production, medium supernatants were removed immediately after the 10-min time point was photographed and stored atDetermination of NO production. NO production was measured in the supernatants from LPS-treated cells after both 24- and 48-h incubations by use of a nitrate/nitrite colorimetric assay kit (Cayman Chemical). Briefly, 80-µl aliquots of supernatant were incubated in nitrate reductase solution containing the necessary cofactors required for conversion of nitrates to nitrites, and combined nitrate and nitrite was measured with Griess reagents, which react with nitrite to form a purple azo compound with an absorbance at 543 nm. Standards were prepared in assay medium with a sample detection limit of 2.5 µM. The cells were resuspended in 0.1 M NaOH after collagenase treatment, and protein content was determined by the Bio-Rad Bradford technique for normalization of nitrite per milligram of protein.
Statistical analysis. Results are expressed as means ± SE unless otherwise noted. For contractility assays, error bars for the control values represent the average SE of triplicate measurements for each experiment. For all assays, statistical analysis was performed with a one-way repeated-measures ANOVA followed by a multiple comparison procedure with the Student-Newman-Keuls method. A value of P < 0.05 was considered significant.
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RESULTS |
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Effects of LPS on rat lung pericyte contractility.
As we expected, incubation of pericytes with LPS significantly reduced
contractility of the pericytes in a concentration-dependent manner.
LPS-induced relaxation (i.e., decreased contraction) was evident by 24 h and still present when tested at 48 h (Fig.
1). At the highest concentration tested
(100 µg/ml), LPS maximally reduced contraction by 49 ± 4 and 42 ± 6% at 24 and 48 h, respectively. LPS-induced relaxation did not occur
with shorter incubations (10 min to 1 h, data not shown).
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Effects of aminoguanidine on LPS-induced relaxation and NO
production.
To determine whether iNOS was involved in mediating the LPS-induced
relaxation, pericytes were treated with LPS and incubated for 24 or 48 h in the presence and absence of aminoguanidine, a noncompetitive
inhibitor of iNOS, and nitrite measurements were taken from the
supernatant medium. NO production was not detectable in the 24-h
contractility assay with the colorimetric assay kit; however, by 48 h,
NO production was evident and significantly upregulated in the
LPS-treated cultures in a concentration-dependent manner (Fig.
2). In the presence of 100 µg/ml LPS, NO
production was increased ~90% (P < 0.05) over the control
(no LPS, no aminoguanidine) value. As expected, aminoguanidine (200 µM) reduced NO levels, significantly decreasing NO synthesis by
~50% (P < 0.05) in control (no LPS) cells and in the
presence of 100 µg/ml LPS. The ability of LPS to induce relaxation in
these assays was not blocked by aminoguanidine (Fig.
3). In fact, in the presence of
aminoguanidine for 48 h, 50 and 100 µg/ml LPS reduced contraction in
the pericytes by 71 ± 4 and 76 ± 4%, respectively, which was
significantly greater than LPS alone. This same effect was also present
after 24 h. In the presence of aminoguanidine, 50 and 100 µg/ml LPS
decreased contraction of pericytes by 66 ± 5 and 71 ± 6%,
respectively, which was also significantly greater than that of LPS
alone.
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Effect of L-NAME and L-NMMA on LPS-induced
relaxation.
To determine whether this increase in LPS-induced relaxation of
pericytes was a general property of NO synthesis inhibitors, the
ability of the NOS inhibitors L-NMMA and L-NAME
to affect LPS-induced relaxation of pericytes was also tested (Fig.
4). As with aminoguanidine, neither of
these NOS inhibitors blocked the LPS-induced relaxation. In the
presence of L-NMMA (200 µM), LPS at concentrations of 50 and 100 µg/ml decreased contraction of the pericytes by 73 ± 7 and
75 ± 7%, respectively, which was significantly greater than LPS
alone at these concentrations. In the presence of L-NAME
(10 µM), LPS decreased contraction in the pericytes by 61 ± 4 and
67 ± 4% at 50 and 100 µg/ml LPS, respectively; however, these
results were not significantly different from LPS alone. Thus even
though LPS stimulated NO production, inhibition of NO synthesis by
three different inhibitors did not have the expected effect of blocking
the LPS-induced relaxation. In addition, there was no significant
effect of LPS or iNOS inhibitors on cell density or viability as
determined by cell counts (data not shown).
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Effect of LPS on contractility in iNOS-deficient lung pericytes.
The ability of LPS to induce relaxation in pericytes lacking the iNOS
gene was also demonstrated. LPS significantly reduced contractility of
the pericytes in a concentration-dependent manner (Fig.
5) similar to that observed in control mice
containing the iNOS gene (data not shown). At the highest concentration
tested (100 µg/ml), LPS maximally reduced contraction by 45 ± 10%,
which is consistent with results obtained in the rat pericyte.
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NO donor effect on pericyte contractility.
Our initial hypothesis was that NO would mediate relaxation of
pericytes; however, the enhanced relaxation of pericytes in the
presence of NO synthesis inhibitors might suggest that instead of
relaxing these cells, NO may actually modulate or mediate contraction in these cells. The ability of NO to contract gastrointestinal smooth
muscle has been demonstrated by others (1). Therefore, to determine
whether NO is capable of either relaxing or contracting lung pericytes,
contractility was measured in the presence of the NO donors SNP, SNAP,
and spermine NONOate. At concentrations shown to produce relaxing
effects in other studies (<104 M; 11, 22, 24),
none of the NO donors had any effect on contractility (Fig.
6). Only spermine NONOate at the highest
concentration tested (10
3 M) caused a significant
reduction in contraction (to 32 ± 4% of control).
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Effects of SNP on LPS-induced relaxation.
Another possible explanation is that NO was only capable of affecting
pericyte tension in the presence of LPS. To test this, SNP was added to
LPS-treated cultures 10 min before release of the gels. As illustrated
in Fig. 7, the relaxation effect of LPS occurred regardless of the presence of SNP (104 M).
There was a nonsignificant trend toward increased relaxation in the
presence of SNP, clearly indicating that this NO donor did not cause
pericyte contraction.
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DISCUSSION |
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This study demonstrates the ability of LPS and the inability of NO alone to induce relaxation of rat lung pericytes. LPS produced relaxation in a concentration-dependent manner that was apparent only after long-term incubation (24 h), suggesting intervening enzyme synthesis. Although LPS upregulated NO synthesis in the contractility assays, the LPS-induced relaxing effect was not blocked in the presence of the NOS inhibitors aminoguanidine, L-NAME, and L-NMMA and was still present in mouse pericytes derived from iNOS-deficient mice. Furthermore, the NO donors SNP, SNAP, and spermine NONOate at concentrations shown to affect contractility in other studies were unable to contract or relax the pericytes in this study. Therefore, although LPS induces lung pericyte relaxation and increases NO production, the two responses appear to be independent.
We considered the possibility that the presence of L-arginine (400 µM) in DMEM may interfere with the ability of competitive NOS inhibitors to effectively inhibit NO production. However, the presence of L-arginine in DMEM has been previously shown to have no effect on phenylephrine-induced contractions in smooth muscle cells or on the ability of L-NAME and LPS (at these same concentrations) to affect phenylephrine-induced contractions (16, 31). Furthermore, because aminoguanidine is a noncompetitive inhibitor of NOS (25), its ability to inhibit induction of the NOS protein is independent of L-arginine (38).
Aminoguanidine is capable of reducing NO in the LPS-treated cultures as well as in control (unstimulated) cultures. The ability of aminoguanidine to reduce NO levels in unstimulated cells suggests the presence of constitutive NOS (cNOS) protein in these cells, which has not been previously described in either pericytes or mesangial cells.
The ability of LPS to induce NO synthesis in this study is consistent with other in vitro studies involving both retinal pericytes and endothelial cells (5, 23) as well as smooth muscle cells (16). In these studies, NO levels were increased ~20-fold; however, in the present study, NO levels are only increased twofold. Because induction of iNOS is responsible for production of excessive amounts of NO compared with cNOS, the ability of LPS to upregulate NO synthesis by only twofold in the present study again suggests the presence of a cNOS protein.
The inability of NOS inhibitors to attenuate LPS-induced relaxation has been previously reported in other tissues. In a study involving mammary artery and saphenous veins (31), LPS attenuated the contractile response to phenylephrine, but L-NAME and L-NMMA were unable to attenuate this response in the saphenous veins, even at concentrations as high as 1 mM. A recent in vitro study (36) involving both endothelium-denuded aortic rings and vascular smooth muscle cells has also demonstrated a hypocontractile effect with LPS, which was only partially restored in the presence of a number of NOS inhibitors. Because aminoguanidine as well as L-NMMA exaggerated the LPS response in the present study, it is possible that these agents have other effects on contractile mechanisms, not previously described, that are independent of their effects on NO synthesis.
The ability of LPS in the present study to induce relaxation in iNOS-deficient mice clearly demonstrates that iNOS is not involved in mediating the LPS response. An in vivo study involving these iNOS-deficient mice (17) has also demonstrated a lack of iNOS involvement in mediating LPS-induced hypotension. In this study, LPS induced hypotension and death even in the complete absence of a functional iNOS gene. Further studies involving these iNOS-deficient mice could be useful in the elucidation of such iNOS-independent mechanisms.
In the present study, NO donors had little effect on contractility.
This is in contrast to other studies involving both retinal pericytes
and smooth muscle cells. In retinal pericytes, SNP at concentrations
that produced no significant effect in the present study was shown to
reduce basal tone and increase cGMP synthesis (11). cGMP is a known
mediator of NO-dependent relaxation in many types of smooth muscle
cells (12). Similarly, SNAP (22) and spermine NONOate (24) at
105 M have been shown to relax smooth muscle cells
from various arteries. The only significant effect of a NO donor
observed in the present study was with 10
3 M
spermine NONOate, which could have been due to nonspecific effects of
the compound at this very high concentration. The inability of the lung
pericyte to respond to NO may be a tissue-specific response and may
indicate the lack of cGMP regulation of the contractile mechanism in
the lung pericyte. The presence of tissue-specific responses has been
previously shown to exist between various capillary beds (32).
In addition to regulating vascular tone, NO has other postulated protective roles including inactivation of oxygen free radicals, prevention of microvascular thrombosis, inhibition of platelet aggregation, and adhesion to endothelial cells as well as stabilization of cell membranes (26). Thus the inability of NO derived from lung pericytes to produce relaxation suggests the presence of another role for this LPS-induced NO.
The presence of a NO-independent /cGMP-independent relaxation pathway has also been shown to exist in cGMP kinase (cGKI)-deficient mice (21). Because NO-mediated relaxation occurs through activation of guanylate cyclase, loss of cGKI abolished NO-cGMP-dependent relaxation of smooth muscle cells; however, cAMP-dependent relaxation was still present. cAMP has previously been demonstrated to relax retinal pericytes through disassembly of stress fibers (14), and prior findings in our laboratory (30) have demonstrated that forskolin, an activator of adenylate cyclase, relaxes lung pericytes. Thus an alternative pathway that could mediate LPS-induced relaxation might involve cAMP.
In conclusion, although NO has been proposed to regulate vascular tone and play an important role in the hypocontractility associated with inflammatory conditions such as shock or sepsis, growing evidence exists that demonstrates the presence of NO-independent mechanisms associated with hypocontractility as well. This study demonstrates that LPS-mediated lung pericyte relaxation occurs by an iNOS-independent mechanism. Further studies of such iNOS-independent contractile responses are needed to better understand the mechanisms involved in regulating capillary hemodynamics.
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ACKNOWLEDGEMENTS |
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We express gratitude to Nic Spanos (Department of Medical Communications, Wayne State University, Detroit, MI) for expertise and work in the development of all photographs for this study and to Pingyang Yu for the generous gift of collagen.
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. P. Steffes, Harper Hospital, Dept. of Surgery, 3990 John R., Detroit, MI 48201 (E-mail: csteffes{at}med.wayne.edu).
Received 19 July 1999; accepted in final form 5 November 1999.
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