© 2001 American Heart Association, Inc.
Cellular Biology |
NAD(P)H Oxidase–Derived Superoxide Mediates Hypercholesterolemia-Induced Leukocyte–Endothelial Cell Adhesion
From the Department of Molecular and Cellular Physiology (K.Y.S., E.C.C., J.M.R., D.N.G.), Louisiana State University Health Sciences Center, Shreveport, La, and Department of Anatomy and Physiology (C.R.R.), College of Veterinary Medicine, Kansas State University, Manhattan, Kan.
Correspondence to D. Neil Granger, PhD, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 E Kings Hwy, Shreveport, LA 71130-3932. E-mail dgrang{at}lsumc.edu
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Abstract |
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Abstract—Experimental animals placed on a high-cholesterol diet for 2 or more weeks exhibit an inflammatory response in postcapillary venules. The aims of this study were to determine (1) whether superoxide mediates the hypercholesterolemia-induced inflammatory response and (2) whether leukocyte and/or vessel wall NAD(P)H oxidase contributes to this response. Intravital videomicroscopy was used to quantify leukocyte–endothelial cell adhesion in cremasteric postcapillary venules of wild-type (WT) mice, CuZn-superoxide dismutase transgenic (SOD TgN) mice, and mice heterozygous (p47phox+/-) or homozygous (p47phox-/-) for NAD(P)H oxidase placed on either a normal diet or high-cholesterol diet (HCD) for 2 weeks. The number of adherent and emigrated leukocytes in postcapillary venules of WT HCD mice was significantly higher than that detected in venules of their normal-diet counterparts. However, the HCD-induced recruitment of adherent and emigrated leukocytes was not observed in SOD TgN mice. Whereas hypercholesterolemic p47phox+/- and WT mice exhibited similar inflammatory responses, p47phox-/- mice did not. Bone marrow chimeras were developed to selectively delete p47phox from either the vessel wall or circulating leukocytes. Whereas WT marrow transplanted into WT mice produced a normal inflammatory response of venules to HCD, chimeric mice with p47phox deficiency in either the vessel wall or leukocytes exhibited an attenuated inflammatory response to HCD that was comparable with that observed in p47phox-/- HCD mice. Our findings indicate that enhanced superoxide production is a critical event that initiates the leukocyte–endothelial cell adhesion in postcapillary venules of HCD mice. NAD(P)H oxidase appears to be an important source of this superoxide.
Key Words: superoxide dismutase • p47phox • microvasculature • leukocyte emigration • chimeras
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypercholesterolemia is a major risk factor for the development of atherosclerosis, a primary cause of mortality in the United States.1 A chronic inflammatory state occurs during atherosclerosis that is characterized by endothelial cell injury, increased endothelial cell adhesion molecule (CAM) expression, and leukocyte infiltration of the vessel wall.2 3 4 5 6 Cytokines such as tumor necrosis factor-
and
-interferon7 8
released from both the normal cellular components of the vessel wall
and infiltrating leukocytes allow this response to continue,
culminating in plaque formation in the vessel wall. Although it is well
established that prolonged hypercholesterolemia can cause these adverse
changes in large arteries such as the aorta and coronary arteries,
there is growing evidence that similar inflammatory changes occur in
the microvasculature during short-term
hypercholesterolemia.9 10
The microvascular responses to hypercholesterolemia are associated with
activation of endothelial cells and leukocytes and manifest as an
impairment of endothelium-dependent relaxation of arterioles, increased
CAM expression, and leukocyte–endothelial cell adhesion in
venules.9 10 11
Furthermore, acute hypercholesterolemia appears to exacerbate the
microvascular injury response to inflammatory stimuli such as
ischemia-reperfusion.12 13 14 The mechanism by which hypercholesterolemia initiates atherosclerotic lesion development is not completely understood, although cholesterol oxidation products such as those found in oxidized LDL cholesterol have been implicated.15 Oxidized LDL promotes endothelial dysfunction and leukocyte chemoattraction in both large vessels and the microcirculation.16 17 18 Increased oxidant stress, resulting from both increased oxygen free radical (OFR) production and decreased nitric oxide (NO) generation, appears to play an important role in the chronic inflammatory responses to hypercholesterolemia and atherosclerosis.19 The proinflammatory effects of OFRs may result from the ability of these reactive species to (1) increase the expression of CAMs on vascular endothelium20 21 ; (2) enhance the production of leukocyte-activating substances (eg, platelet-activating factor, leukotriene B4, and complement 5a); and (3) promote the rolling, firm adhesion and emigration of leukocytes in the vasculature.22 The recognition that OFRs may contribute to the vascular pathology associated with hypercholesterolemia and atherosclerosis has led to an interest in defining the sources of OFRs in these conditions. Potential sources of OFRs include mitochondrial oxidases, xanthine oxidase, lipoxygenase, and NAD(P)H-dependent oxidases.23 24 Although some studies suggest that xanthine oxidase–mediated superoxide production may be one of the earliest vascular responses to hypercholesterolemia,25 26 more recent work has focused on the potential contribution of NAD(P)H oxidase to the enhanced production of OFRs in blood vessels of hypercholesterolemic human subjects and experimental animals.27 28 29 30 31 32
The enzyme NAD(P)H oxidase has been well characterized in phagocytes and, although less is known about its structure and activity in other cells, similarities exist between the phagocytic, endothelial, and vascular smooth muscle NAD(P)H oxidases.33 34 Phagocytic NAD(P)H oxidase consists of both membrane-bound and cytosolic components. The membrane-associated complex consists of 2 subunits, gp91phox and p22phox, whereas at least 2 other subunits (p67phox and p47phox) constitute the cytosolic component. Under basal conditions, these components are kept apart, rendering the enzyme inactive. However, on stimulation, p47phox becomes heavily phosphorylated and, along with p67phox, moves to the cell membrane where it binds the membrane complex, allowing for enzyme activation. The membrane portion of the enzyme then transfers an electron from NADH or, in the case of neutrophils, NADPH to molecular oxygen, forming superoxide.35 Targeted disruption or deletion of the enzyme (or 1 of its 4 subunits) has allowed for an assessment of the contribution of NAD(P)H oxidase to production of superoxide by neutrophils, endothelial cells, and vascular smooth muscle cells.
To date, evidence supporting a role for NAD(P)H oxidase in the hypercholesterolemia-induced vascular dysfunction and in the development of atherosclerosis is conflicting, and this work has largely focused on large arterial vessels.27 29 30 31 A recent study by Guzik et al29 demonstrated an association between vascular superoxide production by NAD(P)H oxidase and the endothelial dysfunction that accompanies hypercholesterolemia in human blood vessels. Whether such an association exists for the microvascular inflammatory changes that occur during hypercholesterolemia has not yet been addressed. Hence, the major aims of this study were (1) to define the role of superoxide, using transgenic (TgN) mice that overexpress the superoxide scavenging enzyme CuZn–superoxide dismutase (SOD), in mediating the leukocyte–endothelial cell adhesion observed in postcapillary venules of acutely hypercholesterolemic mice; (2) to assess the contribution of NAD(P)H oxidase to this hypercholesterolemia-induced inflammatory response using mice that are genetically deficient in the cytosolic p47phox subunit of NAD(P)H oxidase; and (3) to distinguish between the contributions of leukocyte versus vessel wall NAD(P)H oxidase to this response using chimeric mice that are selectively deficient in either leukocyte or vascular wall p47phox.
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Materials and Methods |
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Animals
Wild-type C57BL/6 mice (WT), CD45 congenic B6.SJL-PTPRCPEP/BOY mice (which express CD45.1), and breeder stocks for C57BL/6-TgN[SOD1]3Cje (SOD TgN) mice on a C57BL/6 background were obtained from The Jackson Laboratories (Bar Harbor, ME). The TgN mice were identified by qualitative demonstration of CuZn-SOD using nondenaturing gel electrophoresis followed by nitroblue tetrazolium staining. Breeder stocks for p47phox knockout mice were obtained from Dr Steven Holland (Laboratory of Host Defenses, NIH).36 Heterozygote/homozygote matings were used to produce approximately equal numbers of either heterozygous (p47phox+/-) or homozygous (p47phox-/-) offspring for this study. The genetic identity of animals was determined by PCR analysis of tail clip DNA.
At 9 to 10 weeks of age the mice were placed on either a normal diet (ND) or a high-cholesterol diet (HCD) (Teklad 90221 containing 1.25% cholesterol, 15.8% fat, and 0.125% choline chloride, Harlan Teklad) for 2 weeks (n=5 to 6 per group). Two separate groups of WT mice were used for the SOD and the p47phox sections of the study.
Chimeras
Three combinations of chimeras were used. WT
WT
chimeras were WT animals that received bone marrow cells from CD45
congenic mice, reserving NAD(P)H oxidase function. This resulted in a
significant increase of splenocytes expressing CD45.1 (of donor
origin), from <5% in WT to >90% in the WTWT chimeras, allowing
verification of proper chimera reconstitution, as previously
described.37 CD45 congenic
bone marrow was transplanted into
p47phox-/-
mice, yielding
WTp47phox-/-
chimeras with normal blood cell NAD(P)H oxidase function but a
p47phox-deficient
vessel wall. The
p47phox-/-WT
chimeras with NAD(P)H oxidase–deficient blood cells but normal
vascular wall enzyme activity were produced by transplanting bone
marrow from a
p47phox-/-
mouse into a CD45 congenic mouse.
Bone marrow transfer was performed to create chimeric mice. Briefly, bone marrow cells were isolated from the femurs and tibias of donors and resuspended at 4x107 cells/mL PBS. Recipient mice were irradiated with 2 doses of 500 to 525 rad, 3 hours apart, after which 8x106 donor bone marrow cells in 200 µL of PBS were injected into the femoral vein. The chimeras were kept in autoclaved cages, with 0.2% neomycin drinking water for 2 weeks, after which normal drinking water was used. Six weeks after reconstitution, chimeric mice were placed on HCD for 2 weeks and the cremaster preparation was performed. Flow cytometry was used to verify chimera reconstitution by staining for CD45.1 expression on splenocytes with an FITC-labeled anti-CD45.1 antibody (PharMingen Inc).
Surgical Protocol
The mice were anesthetized with ketamine
hydrochloride (150 mg/kg body weight, IP) and xylazine (7.5 mg/kg body
weight, IP). The right jugular vein was cannulated for administration
of heparinized saline, and the left carotid artery was cannulated for
measurement of systemic arterial pressure. Core body temperature was
maintained at 35±0.5°C. Animal handling procedures were approved by
the Louisiana State University Health Sciences Center Institutional
Animal Care and Use Committee and were in accordance with the
guidelines of the American Physiological Society.
Intravital Microscopy
The cremaster muscle microcirculation was evaluated
in this study because (1) the microvascular and inflammatory responses
to hypercholesterolemia previously described for this tissue are
similar to those reported for other tissues, including rat
mesentery,9 10 12 13 14 38
and (2) unlike the rat, the mouse stores large quantities of fat in the
mesentery, making it technically difficult to locate and visualize
mesenteric venules in both WT and mutant mice in the present study.
Each mouse was placed in a semi-upright position on a plexiglas
microscope stage. The cremaster muscle was isolated and placed across a
viewing pedestal as previously
described.14 38 39
The microcirculation was transilluminated and postcapillary venules
with a wall shear rate (WSR) of 
500 per second and diameters between
20 and 40 µm were observed. The venule with the smallest number of
adherent and emigrated leukocytes at the end of a 30-minute
stabilization period was chosen and 5-minute recordings were made of
the first 100 µm of every 300 µm along the length of the
unstimulated vessel. Venular WSR and leukocyte emigration (number per
field) were measured online at the end of each 5-minute observation
period. Leukocyte rolling velocity
(Vwbc)
and number of rolling (number per minute) and adherent (number per 100
µm) leukocytes were quantified in the cremaster muscle during
playback of videotaped images. The mean value of each parameter in a
single venule was calculated, and comparisons were made between the
experimental groups.
Serum Cholesterol Levels
Serum was frozen for subsequent measurement of
cholesterol levels using a spectrophotometric assay (Sigma Chemical
Co).
White Blood Cell Counts
At the end of each experiment, 25 µL of blood was
mixed with 465 µL of 3% acetic acid and 10 µL of 1% crystal
violet for a white blood cell count using a
hemocytometer.
Statistical Analysis
All values are reported as mean±SEM. ANOVA with the
Scheffé post hoc test was used to compare between groups with
statistical significance set at
P<0.05.
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Results |
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Role of Superoxide in Hypercholesterolemia-Induced Leukocyte–Endothelial Interactions
WT HCD mice had 3-fold higher serum cholesterol levels compared with WT ND mice (Table 1
). Both SOD non-TgN and SOD TgN mice placed on HCD
for 2 weeks exhibited similar increases in serum cholesterol versus
their ND counterparts. Hypercholesterolemia did not significantly alter
white blood cell count (6080±1130.8 versus 5000±1834.4 cells/µL),
leukocyte rolling flux (55.0±22.20 versus 83.0±11.66 leukocytes/min),
or Vwbc
(36.2±11.43 versus 64.2±12.83 µm/second) in the WT mice (ND versus
HCD, respectively) or in the SOD non-TgN or SOD TgN mice (data not
shown), although there was more than a doubling of the number of
adherent leukocytes in postcapillary venules of both WT and SOD non-TgN
mice
(Figure 1). A significant (nearly 4-fold) increase in the
number of emigrated leukocytes was also observed in the WT HCD and SOD
non-TgN HCD groups
(Figure 2) when compared with data from ND mice. However, TgN
mice that overexpress CuZn-SOD (SOD TgN) exhibited a significantly
reduced number of adherent
(Figure 1) and emigrated
(Figure 2) leukocytes when placed on the same HCD diet as the
WT and SOD non-TgN mice. Indeed, leukocyte adherence and emigration in
the SOD TgN HCD mice were similar to values obtained in the
corresponding ND group, suggesting that superoxide plays a major role
in the enhancement of baseline leukocyte–endothelial cell adhesion
associated with hypercholesterolemia. The observed changes in leukocyte
adhesion and emigration were independent of any change in WSR
(Table 1).
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Role of NAD(P)H Oxidase in
Hypercholesterolemia-Induced Leukocyte Adhesion and Emigration
Serum cholesterol levels in
p47phox
heterozygous and knockout mice maintained on ND were comparable
(Table 2). However, placement of
p47phox+/-
and
p47phox-/-
mice on HCD for 2 weeks resulted in a significantly larger increment in
serum cholesterol concentration compared with their WT counterparts.
Nonetheless, the more profound elevation in serum cholesterol was not
accompanied by a more intense inflammatory response in cremasteric
venules. Heterozygous
p47phox
mice maintained on HCD for 2 weeks exhibited comparable elevations in
basal leukocyte adherence
(Figure 3) and emigration
(Figure 4) relative to WT HCD mice. However, the HCD-induced
increases in leukocyte adherence and emigration did not occur in
p47phox-/-
mice
(Figures 3 and 4). In fact, the values of leukocyte adherence
and emigration observed in
p47phox-/-
HCD mice were similar to those measured in mice placed on ND. There
were no significant differences in WSR
(Table 2), white blood cell count, leukocyte rolling flux,
or Vwbc
(data not shown) among any of these experimental
groups.
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Hypercholesterolemia-Induced Inflammatory
Responses in
p47phox-/-
Chimeric Mice
After 2 weeks of HCD, all 3 groups of chimeric mice,
WTWT,
WTp47phox-/-,
and
p47phox-/-WT,
had serum cholesterol levels that were comparable with those of WT HCD
animals but significantly lower than levels found in the
p47phox+/-
and
p47phox-/-
HCD groups
(Table 2
). When venules were studied in these groups, no
differences were noted for WSR
(Table 2), white blood cell count, leukocyte flux, or
Vwbc
(data not shown). WTWT chimeras placed on HCD exhibited high
baseline leukocyte adherence
(Figure 5) and emigration
(Figure 6) analogous to levels observed in WT HCD and
p47phox+/-
HCD groups, which suggests that the process per se of creating the
chimeras did not alter the microvascular inflammatory responses to
hypercholesterolemia. However,
WTp47phox-/-
mice [NAD(P)H oxidase deficiency in the vessel wall but not in the
leukocytes] exhibited significantly lower leukocyte adhesion
(Figure 5) and emigration
(Figure 6) relative to WTWT HCD mice but similar to those
observed in all ND groups and
p47phox-/-
HCD mice. A very similar pattern was noted for the
p47phox-/-WT
chimeras [NAD(P)H oxidase deficiency in leukocytes but not in the
vessel wall]; ie, leukocyte adhesion
(Figure 5) and emigration
(Figure 6) were comparable with values in
p47phox-/-
HCD and
p47phox-/-WT
HCD and ND mice but significantly lower than the responses observed in
the WTWT HCD group. These findings suggest that the presence of
NAD(P)H oxidase in both the vessel wall and circulating leukocytes is
essential for the hypercholesterolemia-induced inflammatory responses
noted in venules of the mouse
cremaster.
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P<0.05 vs WT
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Discussion |
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There is a growing body of evidence that hypercholesterolemia can exert detrimental effects on the cardiovascular system via mechanisms that extend beyond the ability of this lipid to promote the formation of atherosclerotic plaques. The component of the vasculature that bears the brunt of the effects of hypercholesterolemia is the endothelial lining of both large and microscopic branches of the arteries and veins. The endothelial cell activation and/or dysfunction in hypercholesterolemic human subjects and experimental animals is manifested as increased production of OFRs, impaired endothelium-dependent vasodilation, increased endothelial CAM expression, and enhanced leukocyte–endothelial cell adhesion.9 10 11 We have previously demonstrated in C57BL/6 mice that intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 were upregulated in many major organs such as heart, lung, and large intestine, although P-selectin and E-selectin were largely unchanged by HCD for 2 weeks, as in the present study.40 Although several mechanisms have been proposed to explain the endothelial dysfunction associated with hypercholesterolemia, there is considerable circumstantial evidence that implicates OFR production as a key event in the initiation and propagation of the vascular pathology. The results of the present study provide additional support for OFRs as mediators of the microvascular and inflammatory changes elicited by hypercholesterolemia and provide novel insights into the potential source of these OFRs.
Our findings in CuZn-SOD TgN mice, which express 2 to 4 times the normal SOD activity in different tissues,41 support a role for superoxide in mediating the leukocyte–endothelial cell adhesion associated with hypercholesterolemia. We observed that the hypercholesterolemia-induced increments in basal leukocyte adhesion and emigration that are normally observed in postcapillary venules of WT mice (or in SOD non-TgN mice) are largely absent in CuZn-SOD TgN mice. The involvement of superoxide in this hypercholesterolemia-induced inflammatory response is consistent with reports describing (1) enhanced production of superoxide by blood vessels of hypercholesterolemic human subjects and experimental animals,25 26 42 43 (2) the ability of superoxide to induce endothelial CAM expression20 21 and promote leukocyte–endothelial cell adhesion in postcapillary venules,13 16 22 and (3) a protective effect of exogenously administered SOD against impaired endothelium-dependent vasodilation associated with hypercholesterolemia.44 Because superoxide reacts rapidly with NO, the increased superoxide generation by endothelial cells in hypercholesterolemia has been proposed to account for at least a portion of the NO deficit observed in this condition.42 Inasmuch as NO has been shown to serve as an endogenous inhibitor of leukocyte–endothelial cell adhesion,45 46 superoxide-mediated inactivation of NO may represent a major underlying cause of the hypercholesterolemia-induced leukocyte–endothelial cell adhesion.
There are several potential enzymatic sources of superoxide within the vasculature, including xanthine oxidase, NAD(P)H-dependent oxidases, mitochondrial oxidases, and lipoxygenase. Xanthine oxidase has been implicated as a source of superoxide in hypercholesterolemia on the basis of the following 2 observations: (1) plasma xanthine oxidase levels are elevated during hypercholesterolemia,25 and (2) the xanthine oxidase inhibitor oxypurinol appears to blunt the enhanced vessel wall production of superoxide that accompanies experimental hypercholesterolemia.42 47 Another superoxide-generating enzyme that has received recent attention as a mediator of the vascular dysfunction associated with hypercholesterolemia is NAD(P)H oxidase. There are several studies that support this possibility. For example, a strong association between vascular superoxide production by NAD(P)H oxidase and the endothelial dysfunction that accompanies hypercholesterolemia in human blood vessels was recently demonstrated.29 It has also been shown that rabbits placed on HCD for 8 weeks exhibit early aortic lesion development associated with an increased production of superoxide that is only partially reduced by oxypurinol but completely inhibited by diphenyleneiodonium, a potent inhibitor of flavin-containing enzymes such as NAD(P)H oxidase.48 There are also reports that do not support a role for NAD(P)H oxidase in hypercholesterolemia-induced vascular pathology, including atherosclerotic lesion development. For example, when apolipoprotein E-/- mice (which spontaneously develop hypercholesterolemia and atherosclerotic lesions) were crossbred with p47phox-/- mice30 or gp91phox-/- mice,31 deleting the NAD(P)H oxidase enzyme did not alter the macrophage infiltration or lesion development in arteries that was normally observed in apolipoprotein E-/- mice.
To address the potential contribution of NAD(P)H oxidase to the superoxide-mediated leukocyte–endothelial cell adhesion associated with hypercholesterolemia, we placed p47phox-/- mice on HCD, like their WT counterparts and heterozygous (p47phox+/-) siblings (with fully functional NAD(P)H oxidase activity), and compared the microvascular responses. The results of these experiments clearly demonstrate an ablation of the hypercholesterolemia-induced inflammatory responses in p47phox-/- mice. The fact that the attenuated inflammatory responses observed in p47phox-/- mice were of comparable magnitude to the attenuation noted in CuZn-SOD TgN mice suggests that NAD(P)H oxidase is the major source of superoxide that mediates these hypercholesterolemia-induced microvascular alterations.
NAD(P)H oxidase is present in many cell types,
including phagocytes, B lymphocytes, endothelial cells, and vascular
smooth muscle cells.35 This
raises the possibility that the cellular source of the NAD(P)H oxidase
that is generating superoxide in response to hypercholesterolemia is
either the venular endothelial cell, circulating leukocyte, or both. We
attempted to determine the relative contributions of
leukocyte-versus-vessel wall–associated NAD(P)H oxidase to the
hypercholesterolemia-induced inflammatory responses using bone marrow
chimeric mice. By transplanting marrow harvested from
p47phox-/-
mice into WT mice or vice versa, we produced mice with NAD(P)H oxidase
deficiency in leukocytes but not in the vessel wall or vice versa, ie,
NAD(P)H oxidase deficiency in the vessel wall but not in the leukocyte.
Control chimeric animals (WT marrow transplanted into WT mice)
exhibited an inflammatory response to HCD comparable with that observed
in WT mice, indicating that the irradiation and marrow transfer
procedures per se do not interfere with the leukocyte adhesion and
emigration responses elicited by HCD. However, the data obtained with
WTp47phox-/-
and
p47phox-/-WT
chimeras indicate that deficiency of NAD(P)H oxidase in either the
circulating leukocytes or the vessel wall attenuates the HCD-induced
leukocyte adhesion and emigration to an extent comparable with that
observed in
p47phox-/-
mice. This observation suggests that both the leukocyte- and vessel
wall–associated forms of NAD(P)H oxidase are necessary for the
superoxide-mediated, HCD-induced leukocyte–endothelial cell adhesion.
Although a definitive explanation for this dependence of the
inflammatory response on both sources of NAD(P)H oxidase is not readily
available, it may reflect a HCD-initiated process whereby NAD(P)H
oxidase–derived superoxide from one cell type (eg, leukocytes) is
required to activate the same enzyme in the other cell population (eg,
endothelial cells). Hence, if NAD(P)H oxidase–mediated formation of
superoxide in endothelial cells is necessary to induce the
leukocyte–endothelial cell adhesion and superoxide produced by the
leukocyte is responsible for activating the endothelial enzyme, then
inhibition or deletion of either source of NAD(P)H oxidase should
afford a comparable level of protection against HCD. This does not
exclude the likely possibility of other inflammatory mediators such as
cytokines or CAMs from participating in this response, nor does it
exclude the possibility that the superoxide derived from either cell
type influences the expression of important inflammatory proteins other
than NAD(P)H oxidase.
An interesting and potentially important observation in this study was the significantly greater increments in plasma cholesterol levels that were observed in the p47phox+/- and p47phox-/- mice placed on HCD versus WT animals. Because the further elevation in plasma cholesterol levels in the p47phox+/- HCD group did not result in a correspondingly larger increment in leukocyte adhesion/emigration and the p47phox deficiency was associated with inhibition of the inflammatory response, the data suggest that the further increments in plasma cholesterol exerted little if any direct influence on the HCD-induced responses. This is consistent with other reports that demonstrate no further amplification of an inflammatory response in microvessels once a threshold serum cholesterol level is reached.14 38 Nonetheless, the exaggerated elevations in plasma cholesterol noted in p47phox+/- and p47phox-/- mice placed on HCD suggest that manipulation of this gene may affect cholesterol metabolism and/or its distribution.
Although much of the work in the area of atherosclerosis research has focused on plaque formation in and dysfunction of large arteries, relatively little attention has been devoted to the potentially important and rather profound inflammatory phenotype in venules that precedes the appearance of large artery plaques and lesions. Nonetheless, there appear to be several shared mechanistic features between the early venular responses and the later large vessel dysfunction, including increased superoxide formation/bioavailability. Although the relevance of an early, superoxide-dependent inflammatory phenotype in venules to the later plaque formation and vessel dysfunction in large arteries remains unclear, the venular response may be equally important if it predisposes the microvasculature to the injurious effects of disease processes that are associated with an acute inflammatory response, such as ischemia/reperfusion.12 13 14 Hence, hypercholesterolemia may increase the risk for cardiovascular disease not only by rendering tissues more likely to experience an ischemic episode (due to vessel obstruction with plaques) but also by exacerbating the inflammatory and tissue injury responses to a given ischemic insult.
In conclusion, the results of this study suggest that hypercholesterolemia induces a superoxide-dependent adhesion and emigration of leukocytes in postcapillary venules. The major source of the superoxide that mediates the HCD-induced inflammatory response is the enzyme NAD(P)H oxidase. Both leukocyte- and vessel wall–associated forms of NAD(P)H oxidase appear to be necessary for the superoxide-mediated, HCD-induced leukocyte–endothelial cell adhesion.
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Acknowledgments |
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This study was supported by a grant from the National Heart, Lung, and Blood Institute (HL26441).
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Footnotes |
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Original received November 21, 2000; revision received January 5, 2001; accepted January 18, 2001.
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References |
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