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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Vasc Surg. 2010 Dec 15;53(3):754–763. doi: 10.1016/j.jvs.2010.09.038

Early Hypercholesterolemia Contributes to Vasomotor Dysfunction and Injury Associated Atherogenesis that can be Inhibited by Nitric Oxide

Kathleen G Raman *,*, Robin E Gandley †,*, Jennifer D Rohland †, Mazen S Zenati *, Edith Tzeng *
PMCID: PMC3052950  NIHMSID: NIHMS242652  PMID: 21163611

Abstract

Objective

Atherosclerosis results in vasomotor dysfunction, in part, through impairment of nitric oxide (NO) dependent vasodilation. It is unclear whether blood vessels are dysfunctional in an early environment of hypercholesterolemia alone and if this contributes to the vascular injury response. We hypothesize that early hypercholesterolemia, prior to gross vascular changes, contributes to vasomotor dysfunction and the vascular injury response. The efficacy of NO therapy to protect against the injury response in this setting was also assessed.

Methods

The effect of oxidized LDL (oxLDL) and inducible NO synthase (iNOS) gene transfer on rat aortic smooth muscle cell (SMC) proliferation was measured with 3H-thymidine incorporation. Carotid arteries (CCA) from wild-type C57BL6 (WT or C57) and ApoE deficient (ApoE KO) mice fed normal or Western diets for 6–8 weeks were tested for vasomotor function using an arteriograph system. Studies were repeated after CCA injury. The effect of iNOS gene transfer on morphometry by histology and vasomotor responses in injured CCAs in ApoE KO was aexamined.

Results

OxLDL increased SMC proliferation by >50%. In SMC expressing iNOS, NO production was unaffected by oxLDL and reduced oxLDL mediated SMC proliferation. Endothelium dependent vasorelaxation was reduced in uninjured CCAs from ApoE KO and C57 mice on the Western vs. normal diet (ApoE 39 ± 2 vs 55 ± 13%; C57 50 ±13 vs 76 ± 5, P<.001) and was increased with longer durations of hypercholesterolemia. Endothelium-dependent and independent vasodilator responses were severely disrupted in C57 and ApoE KO mice 2 wks following CCA injury but both recovered by 4 wks. CCA injury in ApoE KO mice resulted in the formation of atheromatous lesions while C57 mice showed no change (intima 27795 ± 1829 vs 237 ± 28 μm2; media 46306 ± 2448 vs 11714 ± 392 μm2, respectively; P<.001). This structural change in the ApoE KO reduced distensibility and increased stiffness. Finally, iNOS gene transfer to injured CCA in ApoE KO mice dramatically reduced atheromatous neointimal lesion formation.

Conclusions

Early hypercholesterolemia impairs endothelial function, with severity being related to duration and magnitude of hypercholesterolemia. Severe hypercholesterolemia leads to atheromatous lesion formation following injury and stresses the role of vascular injury in atherogenesis and suggests different mechanisms are involved in endothelial dysfunction and the injury response. Despite these changes, iNOS gene transfer still effectively inhibits atheroma formation. These findings support early correction of hypercholesterolemia and emphasize the potential role for NO based therapies in disease states.

INTRODUCTION

Atherosclerosis is a leading cause of morbidity and mortality(1). It involves changes in vascular architecture and composition that render vessels susceptible to complications such as stenosis and thrombosis. Early changes in the arterial wall include fatty streaks composed of lipid filled inflammatory cells and extracellular lipids. These lesions are common in early life and, while some progress to atherosclerotic lesions, others remain unchanged or resolve in adulthood (2).

Factors that determine the fate of these early lesions are unknown but the endothelium appears to be involved. Endothelial dysfunction is reported to be a systemic disease and may predict cardiovascular risk (3–5). It is a systemic marker of arterial damage from the risk factors of cardiovascular disease and reflects the body's ability to repair or compensate for this damage. Impaired vasomotor function in patients with cardiovascular comorbidities is linked to impaired endothelial NO production or reduced NO bioavailability (3,4,6,7) which results in vasoconstriction and the generation of reactive nitrogen species (4,8,9) that produce lipid peroxidation and cell injury.

Vascular injury in mouse models of hypercholesterolemia or hyperlipidemia enhances intimal hyperplasia (IH) (10–12). Enhanced inflammation appears to be associated with the greater IH observed in hypercholesterolemic ApoE KO mice (11). It is also established that vascular injury resulting in endothelial damage and loss of NO contributes to IH (13,14). We and others have reported that IH in injured normal arteries is markedly reduced by inducible NO synthase (iNOS) gene transfer to the site of injury (15–17). Because lipids can consume NO (8,9), the efficacy of iNOS gene transfer in the setting of atherosclerosis may be significantly attenuated.

In this report, we hypothesize that early hypercholesterolemia, prior to gross vascular changes, contributes to vasomotor dysfunction and the vascular injury response (18,19). To address this, we examined the effect of diet/hypercholesterolemia on vasomotor responses and on the response to vascular in mice. Because of the poorly defined role of NO in atherogenesis, we evaluated the ability of iNOS gene transfer to inhibit IH in hypercholesterolemia.

MATERIALS AND METHODS

Experimental Animals

Animals were housed in an Association for the Assessment and Accreditation of Laboratory Animal Care -accredited animal facility. Male Apolipoprotein E deficient mice (ApoE KO) and control C57BL6J (C57) mice (Jackson Labs, Bar Harbor, ME) at 6 wks of age were fed a chow or a Western diet (21% fat, 0.2% cholesterol by weight; Harlan Teklad, Madison, WI) for 6–8 wks total (18,19).

Carotid artery injury model

All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh. Mice were anesthetized using Nembutal (50mg/kg IP) and isofluorane (Abbott Labs, Chicago, IL). Right carotid injury was performed as described (20). Briefly, an 0.018 wire was inserted into the CCA, rotated 3 full turns to denude the endothelium, and then removed. The left CCA served as the uninjured control.

Adenoviral vectors

An E1-, E3- deleted adenovirus carrying the human iNOS cDNA (AdiNOS) was constructed as described (16). The control, AdlacZ, carried the ß-galactosidase gene. Following carotid injury, adenovirus (1 × 109 PFU/ml) was applied topically to the adventitia of the CCA using Poloxamer 407 (BASF Corp, Mount Olive, NJ) gel (100 μL) to enhance retention and gene transfer (21). Gene transfer efficiency was estimated in AdlacZ vessels using X-gal staining at day 3.

Morphometric analysis

Mice were euthanized 28 days post-injury and and serum was stored for analysis. For each mouse, the heart was perfused with PBS and then 2% paraformaldehyde. CCAs were excised and fixed, sectioned, and stained with H&E as described (16). Intimal and medial areas were quantified (MetaMorph®, Downington, PA) for 8 sections/vessel. Sections were also examined for elastin autofluorescence and under polarized light for lipid desposition.

SMC proliferation

Aortic SMC were cultured from mouse aortae as described (22) and used at passages 3–6. Growth-arrested SMCs were cultured with 10% FBS + 5 μCi/mL 3H-thymidine (Perkin Elmer, Boston, MA) as described (23). For some experiments, cells were first infected with AdiNOS or AdLacZ at a multiplicity of infection (MOI) of 10 for 1 hr and serum starved for 24 hrs. 3H-thymidine incorporation was quantified at 24 hrs. Tetrahydrobiopterin (BH4) (10μmol/L) was added to optimize iNOS activity. The effect of oxidized LDL (oxLDL, Sigma, St. Louis, MO) (24) on SMC proliferation was also evaluated. NO synthesis was quantified by the Griess reaction (16)

Cholesterol analysis

Serum cholesterol was measured in fasted mice using a chromogenic assay (Sigma) and was calculated against standards.

Isolated Arteriograph Experiments

CCAs were collected from euthanized mice and placed in cold HEPES-buffered physiologic saline solution (HPSS) (25). Vessels were mounted on microcannulae in a pressurized arteriograph (Living Systems, Burlington VT) at 37°C, pH 7.4with a video camera and dimension analyzing system (Living Systems) to record lumen diameter and wall thickness (26). A conditioning stretch was performed by increasing the intraluminal pressure from 60 to 100 mm Hg and returning to 60 mm Hg followed by 15 min of equilibration. CCAs were preconstricted with the thromboxane analog U46619 (23) and treated with methacholine (ME; 10−8 – 10−6 mol/L) or sodium nitroprusside (SNP; 10−9 – 10−4 mol/L).

Myogenic tone

An approach modified from MacPherson et al was utilized to measure myogenic reactivity (27,28). Intraluminal pressure was decreased to 20 mm Hg for 10 min and then increased from 20–120 mm Hg at 4–6 min intervals. Smooth muscle was inactivated with calcium-free HPSS, 10−4 mol/L papaverine and 10−4 mol/L EGTA and passive luminal diameter and wall thickness were measured at pressures from 0–150 mm Hg. % tone = [(Dr − DPSS)/Dr] × 100, where Dr is the relaxed diameter in the calcium-free buffer and DPSS is the diameter in HPSS.

Calculations

Distensibility is the Δdiameter/Δpressure in arteries with inactivated smooth muscle. To obtain the relative change in diameter, the diameter at each pressure was normalized to the initial diameter at 5 mm Hg. The slopes of the pressure-diameter curves were used to compare distensibility. Circumferential stress is the force exerted on the vascular wall per unit of tissue [stress = (P × D) /2T; P is the transmural pressure in mNewtons per mm2 (1 mmHg = 0.133 mN/mm2), D=diameter, and T=wall thickness]. Circumferential strain is the response of an artery to force. Strain is (Df – D0)/D0 where D0 is the initial diameter at 5 mmHg and Df is the diameter at the new pressure. Stress was calculated for specified strains.

Statistical analysis

Data are expressed as mean ± SE. Student's t-test was used for pair-wise comparisons of parametric data. All data were first analyzed by one-or two-factor ANOVA. If significant, the means were compared with the level of significance for each test adjusted by the Bonferroni method. Two-way repeated measures ANOVA was used to compare changes in diameter at different pressures between groups. P<0.05 indicated significance.

RESULTS

OxLDL stimulates SMC proliferation

To examine the effect of an atherogenic environment on SMC proliferation, we treated SMC with oxLDL in vitro. SMC from ApoE KO and C57 mice exhibited similar proliferation rates. OxLDL (50 μg/mL) increased proliferation by >50% in both cell types (P<0.001, Fig. 1A) vs. cells treated with LDL. iNOS expression increased NO release (Fig. 1B) while AdlacZ treated cells produced little NO. zOxLDL did not alter NO production by AdiNOS treated cells. AdiNOS infection of ApoE KO and C57 SMC reduced proliferation by 65–70% vs. AdlacZ treated cells. In the presence of oxLDL, AdiNOS still reduced proliferation by over 70%.

Figure 1.

Figure 1

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OxLDL stimulates SMC proliferation in vitro. SMC were cultured and oxLDL (50 μg/mL) in growth medium was added to the SMC and proliferation was quantified using 3H-thymidine incorporation over 24 hrs (A). Proliferation was expressed as % of SMC cultured in 10% serum. SMC were also treated with AdiNOS or AdlacZ. The effect of NO with and without oxLDL on SMC proliferation was also assessed. Griess reaction was performed to quantify NO production by the SMCs (B). *P<.001 vs. all other groups (except AdiNOS + oxLDL vs AdlacZ + oxLDL), †P<.05 vs. ApoE KO under the same conditions, ‡P<.001 vs. all other groups.

Effect of diet on scholesterol/triglyceride levels

On chow, ApoE KO mice had a 7-fold greater cholesterol level than C57. C57 on a Western diet had a doubling of cholesterol (Table 1, P=0.002) but was significantly lower than ApoE KO on chow. ApoE KO on a Western diet had markedly elevated cholesterol levels (P<0.001 vs. all other groups). Triglycerides were similar in all groups. Gene transfer of iNOS or lacZ did not affect cholesterol or triglyceride levels in ApoE KO.

Table 1.

Serum cholesterol and triglyceride concentrations

Mouse/Diet N=8 Cholesterol (mg/dL) P value Triglycerides (mg/dL) P value
C57/chow 65.6 ± 11.4 -- 8.4 ± 1.2 --
C57/Western 150.4 ± 13.4 0.002* 9.3 ± 2.1 NS
ApoE/chow 472.0 ± 15.4 <0.001*† 8.6 ± 1.4 NS
ApoE/Western 1276.4 ± 43.9 <0.001*† 9.3 ± 1.4 NS
ApoE/Western AdlacZ 1247.3 ± 60.0 <0.001*‡ 10.5 ± 1.5 NS
ApoE/Western AdiNOS 1223.7 ± 70.5 <0.001*‡ 10.4 ± 1.4 NS
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Mean ± SD, n=8/group

*

versus C57/chow

†

versus all other groups

‡

versus C57/chow, C57/Western, ApoE/chow

Effect of diet on the arterial injury response

Vascular injury contributes to atherosclerotic plaque formation. We examined the effect of early hypercholesterolemia on arterial injury. Carotid injury in C57 minimally altered the arterial wall by 28 days post-injury (Fig. 2A,C,G), remaining similar to uninjured CCA (not shown). ApoE KO, in contrast, had a significant intima (Fig. 2B,D,G; 27795 ± 1829 vs. 237 ± 28 μm2, P<0.001) with media (46306 ± 2448 vs. 11714 ± 392 μm2, P<0.001) response. Polarized light (Fig. 2E,F) showed lipid deposition throughout the lesions in ApoE KO vessels, resembling atheromas. C57 vessels had little lipid accumulation. Contralateral uninjured CCAs from ApoE KO had no lipid accumulation despite a high fat diet for 8 wks (data not shown). Thus, early hypercholesterolemia adversely affects the injury response but only in the setting of severe elevations in cholesterol.

Figure 2.

Figure 2

Figure 2

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The vascular injury response is increased in ApoE KO mice compared with C57 mice. Carotid injury was created with a wire and arteries were collected 4 wks later. Vessels were fixed, sectioned and stained with H&E and imaged at 200x. A) C57 artery with H&E; B) ApoE KO artery with H&E; C) C57 artery under auto-fluorescence; D) ApoE KO artery under autofluorescence; E) C57 artery under polarized light; F) ApoE KO artery under polarized light; G) quantification of intimal and medial arteries (mean of 8 sections per artery and 8 mice per group, error bars represent SEM), *P<.001 vs. C57.

The effect of diet and injury on endothelium dependent vasorelaxation

Vasorelaxation of CCAs from ApoE KO and C57 mice fed a chow or a Western diet for 8 wks was examined. Endothelial dependent relaxation to methacholine was reduced in intact CCA from ApoE KO on a Western diet vs. CCAs from ApoE KO on chow (39 ± 2 vs. 55 ± 13%, P<0.001) (Fig. 3A). Similar dietary effects were seen in C57 where the Western diet reduced vasorelaxation vs. chow (50 ± 13 vs. 76 ± 5%, P<0.001). Response to methacholine was worse in ApoE KO vs. C57 (P=0.002) within each diet group.

Figure 3.

Figure 3

Figure 3

Figure 3

Figure 3

Figure 3

Figure 3

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Vasorelaxation responses of isolated carotid arteries are significantly reduced in A) intact arteries from mice on Western diet for 8 weeks [open circles and squares; * WT normal diet vs. APO E KO Western, P≤.04; # WT normal diet vs. WT Western, P≤.03; ^ APO E KO normal diet vs. APO E KO Western, P=.021; + WT normal diet vs. APO E KO normal diet, P=.022] or B) in the contralateral injured arteries from the same animals [solid circles and squares; * WT normal diet vs. APO E KO Western, P<.001; # APO E KO normal diet vs. APO E KO Western, P<.001; ^ WT Western vs. APO E KO Western, P≤.02; + APO E KO normal diet vs. WT Western, P≤.02; % WT normal diet vs. WT Western, P=.01]. The influence of diet (8 wks) and injury (4 wks) on arteries from WT mice is represented in C [* normal diet INT vs. Western INJ, P≤.02; + normal diet INT vs. Western INT, P≤.03], and in arteries from APO E KO mice in D [*normal diet INJ vs. Western INJ, P<.001; + normal diet INJ vs. Western INT P≤.009; − normal diet INT vs. Western INJ, P<.001; ^ normal diet INT vs. Western INT, P≤.03; # normal diet INJ vs. normal diet INT, P=.029]. At 2 weeks post-injury, injured carotid arteries from APO E KO mice on Western diet had E) very little endothelial mediated relaxation (solid circles,* P≤.001) and had F) reduced smooth muscle mediated relaxation in response to a nitric oxide donor (solid circles, * P≤.001). At 4 wks post-injury relaxation to NO donor has recovered (B–D).

At 4 wks after injury, endothelium dependent relaxation in ApoE KO and C57 on a Western diet resembled intact vessels from the same strains (ApoE KO 39 ± 2 vs 34 ± 3%; C57 51 ± 13 vs 51 ± 4%; intact vs injured, respectively) (Fig. 3A–D), reflecting endothelial healing. Chow fed mice had similar responses in intact and injured CCA (ApoE KO 55 ± 13 vs. 66 ± 5%; C57 76 ± 5 vs. 63 ± 5%). Because vessels appeared to be fully healed by 4 wks, endothelial function was examined 2 wks post-injury in ApoE KO on a Western diet. These vessels had marked impairment in endothelium mediated vasorelaxation vs. intact arteries (Fig. 3E) and vessels 4 wks post-injury (7 ± 14% at 2 wks vs. 34 ± 3% at 4 wks; P<0.02)(Fig. 3B–E). At 2 wks post-injury, arteries from ApoE KO on a Western diet exhibited impaired endothelium-independent relaxation to SNP vs. intact arteries (19 ± 9 vs. 75 ± 20%; P<0.001)(Fig. 3F) or arteries 4 wks post-injury (96 ± 12%). The response to SNP 4 wks post-injury resembled uninjured vessels regardless of dietary influences (data not shown). Methacholine responses were reduced in intact arteries from ApoE KO after 8 wks vs. 6 wks on a Western diet (39 ± 2 vs.70 ± 15%, P=0.03) (Fig. 3A,C–E). These findings indicate that endothelial healing does occur following vascular injury in the setting of hypercholesterolemia and extreme changes to the arterial architecture. However, endothelial function is more dramatically affected by duration of hypercholesterolemia.

Myogenic tone and passive mechanical properties

The ability of blood vessels to actively contract to increases in intraluminal pressure is defined as myogenic tone. The tone in intact CCA from ApoE KO on Western or chow diet was significantly greater vs. injured arteries (8 vs. 2.5% on Western diet, P<0.001; 6 vs. 1%, on chow diet, P<0.003; Fig. 4A). C57 had similar responses where tone was reduced from 7% to 2% with injury (P<0.001). Unlike endothelial dependent responses, myogenic tone was not improved 4 wks after injury vs. 2 wks (data not shown).

Figure 4.

Figure 4

Figure 4

Figure 4

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Active and passive properties of isolated carotid arteries. A) Active contractility in response to pressure stimuli (myogenic tone) was lost in injured arteries from APO E KO mice regardless of diet (solid symbols). Myogenic tone was lost in all injured arteries at both 2 and 4 wks post-injury (data not shown for 2 wks or WT mice). [*Western Intact vs. normal diet Injured, P≤.03; # Western Intact vs. Western Injured, P≤.001; ^ normal diet Intact vs. Western Injured, P≤.05; + normal diet Intact vs. normal diet Injured, P≤.02; % Western Intact vs. normal diet Intact, P=.022]. B) Distensibility was increased in the injured carotid arteries at 2 wks post-surgery (solid black squares), while the injured arteries were less distensible than intact carotid arteries regardless of diet at 4 wks post-injury in APO E KO mice [*Western 2 WK Injured vs. Western Intact, P<.034; ^ Western 2 WK Injured vs. Western 2 WK Intact, P<.009; # Western 2 WK Injured vs. Western Injured, P<.009; % Western 2 WK Intact vs. Western Injured, P<.001]. C) Stress-strain calculations showed that injured arteries after 4 wks of recovery experienced greater amounts of stress per given strain [* APO E KO Western Injured vs. WT Western Intact, P≤.03; # APO E KO Western Injured vs. APO E KO Western Intact, P≤.04; ^ APO E KO Western Injured vs. WT Western Injured, P≤.02, + WT Western Intact vs. WT Western Injured, P≤.03].

Pressure transduction through the arterial wall to stimulate contraction is dependent on wall composition (29). We measured the passive mechanical properties of intact and injured CCAs. At 4 wks post-injury, maximal CCA diameters were reduced in injured vs. uninjured arteries from ApoE KO on chow (538 ± 39 vs. 629 ± 37 μm, P=0.015) or Western diet (540 ± 30 vs. 615 ± 26 μm, P=0.016). Similar reductions were seen in C57 mice. Diet did not alter these responses in either mouse strain. Arteries 4 wks post-injury were less distensible than uninjured arteries (C57 P<0.001, ApoE KO P<0.001). Distensibility was not influenced by diet (Fig. 4B) but was significantly reduced in injured vs. uninjured arteries in all groups at 4 wks (0.71 ± 0.05 vs. 0.89 ± 0.02, P=0.003). In contrast, at 2 wks, injured arteries from ApoE KO on a Western diet were more distensible than uninjured ones, consistent with significant medial injury.

Wall thickness was increased at 4 wks post-injury in ApoE KO on Western diet vs. uninjured arteries (52 ± 6 vs. 33 ± 1 μm at 120 mmHg, P=0.02) with similar changes in injured arteries from C57 regardless of diet. The stress-strain characteristics of arteries take into account changes in vessel wall thickness. The stress-strain relationships were identical for the vessels from ApoE KO and C57 on a Western diet. Four wks after injury, however, the arteries from ApoE KO demonstrated a leftward shift at larger strains with reduced changes in vessel cross-sectional area for a given stress vs. intact vessels from either ApoE KO or C57 (Fig. 4C, P<0.001) as well as injured vessels from C57 (P<0.03). This indicates that the arterial wall had become stiffer and is consistent with the architectural changes observed at 4 wks post-injury in the ApoE KO vessels where there were atheromatous intimal/medial lesions.

Effect of NO on the vascular injury response

To determine if NO exhibits therapeutic actions in hypercholesterolemia, iNOS gene transfer was performed in ApoE KO on a Western diet (Fig. 5). X-gal staining of AdlacZ treated CCAs revealed transgene expression throughout the arterial wall (Fig. 5E,F). At 28 days following injury, iNOS gene transfer reduced both intimal (72%, P=0.04) and medial (73%, P=0.001) areas (Fig. 5G). AdlacZ treated CCA developed intimal and medial lesions (Fig. 5A,C) containing aggregates of cells and matrix. Polarized light revealed lipid accumulation in these lesions (data not shown). iNOS gene transfer dramatically inhibited this accumulation of intimal and medial cells (Fig. 5B,D). The effect of NO on vasomotor recovery was examined at the 2 wk time point where marked endothelium-dependent and independent vasodilator dysfunction. However, there was little vasorelaxation to methacholine in AdiNOS or AdlacZ treated arteries detected (Fig. 6).

Figure 5.

Figure 5

Figure 5

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iNOS gene transfer inhibits intimal and medial hyperplasia in injured CCAs. ApoE KO mice underwent wire injury of the carotid artery followed by adventitial gene transfer with AdiNOS or AdlacZ. CCAs were isolated 4 wks later, sectioned and stained and imaged at 200x. A) AdlacZ vessel stained with H&E; B) AdiNOS vessel stained with H&E; C) AdlacZ vessel under auto-fluorescence; D) AdiNOS vessel under auto-fluorescence; E) AdiNOS vessel stained for β-galactosidase with X-gal; F) AdlacZ stained with X-gal; G) morphometric analysis of intimal and medial areas (8 semi-serial sections/vessel, 8 animals per group, *P<0.001 vs. AdlacZ)

Figure 6.

Figure 6

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iNOS gene transfer does not accelerate vasomotor recovery following injury. ApoE KO mice on Western diet underwent wire injury of the CCA followed by adventitial gene transfer with AdiNOS or AdlacZ. CCAs were isolated 2 wks later for vasorelaxation to methacoline studies and data are presented in comparison to untransfected intact and injured arteries from ApoE KO mice at same time point. Methacholine response curve shows poor endothelium dependent vasorelaxation in all injury groups.

DISCUSSION

Atherosclerosis affects vascular properties by limiting the flow lumen, rupturing and inducing thrombosis, and promoting inflammation. However, the effect of diet and hypercholesterolemia prior to plaque formation on vasomotor function and the vascular injury response is not well established. In this study, CCAs from 14–16 wk old ApoE KO and C57 mice showed no evidence of atherosclerotic plaque regardless of the diet or serum cholesterol levels but hypercholesterolemia in both strains of mice impaired endothelium dependent vasodilation. Endothelial dysfunction correlated with the degree of hypercholesterolemia from dietary or genetic factors. This dietary effect was cumulative over time with greater endothelial dysfunction in animals on a Western diet for 8 wks vs. 6 wks. These findings suggest that diet plays a key role in endothelial dysfunction early after onset of hypercholesterolemia and well before plaque formation. It is the consensus that early changes in endothelium dependent vasodilation are observed in patients with risk factors for atherosclerosis such as hypertension, DM, and hypercholesterolemia (4). Prior reports have also identified similar dietary influences on endothelium-dependent vasodilation (7,18,30,31). However, our studies were performed after only 6–8 wks of diet and supports early correction of even mild levels of hypercholesterolemia to prevent vascular sequelae.

We did not investigate the local changes induced by hypercholesterolemia but the evidence supports that early changes in the redox equilibrium in the arterial wall can sequester endothelium derived NO. d'Uscio et al (30) reported that endothelium-dependent vasodilation was restored in ApoE KO with superoxide dismutase. These experiments were performed in mice fed a Western diet for over 26 wks and the reactive oxygen species (ROS) production localized to the atheromas. Ohara et al (31) reported increased endothelial superoxide production in aortic rings from rabbits fed a high cholesterol diet for 1 month. Our data suggest that changes in arterial ROS production may occur very early in hypercholesterolemia.

The greatest impact of hypercholesterolemia was detected following vascular injury. ApoE KO CCA developed marked medial and intimal hyperplasia and atheroma formation following injury. Our findings are similar to those of Matter et al (11) who reported minimal IH in C57 mice but marked intimal and medial changes in ApoE KO following carotid injury. These changes were further enhanced with a high cholesterol diet, supporting the contribution of hypercholesterolemia. There was a similar gradation in inflammatory changes between the groups as indicated by VCAM-1 expression and inflammatory cell infiltration. The correlation between cholesterol levels and the degree of inflammation was supported by Kaufmann et al (23) who showed incremental increases in VCAM in arteries of C57 mice on chow, C57 on a Western diet, ApoE KO on chow, and the greatest in ApoE KO on a Western diet. The response in ApoE KO involved proliferation and lipid uptake in intimal/medial cells. In our studies, a mildly elevated cholesterol in C57 mice on the Western diet yielded minimal IH and lipid deposition following injury. This suggests that the increased inflammation associated with marked hypercholesterolemia in the ApoE KO mice (11,23) plays a greater role in IH and lipid deposition than the resultant endothelial dysfunction. These findings also indicate that the vascular injury response in hypercholesterolemia is strikingly different from normal animals, illustrating their poor predictive value in testing potential therapies.

Vascular injury underlies the pathogenesis of atherosclerosis. Thus, we examined the impact of injury on endothelial function and its recovery. Arteries from ApoE KO mice 2 wks after injury showed reduced endothelium-dependent and independent relaxation, indicating medial injury. While the reduced methacholine response would suggest endothelial damage, the hyporesponsive medial SMC to SNP indicates medial damage is prevalent at this time. By 4 wks, both endothelium-dependent and -independent responses returned to baseline suggesting healing of both the endothelium and the SMC. We did not perform imaging to look at reendothelialization but the recovery of methacholine responses indicates this did occur. The injured Apo E KO vessels recovered and resembled the contralateral uninjured artery which was unexpected given the dramatic structural change in the arterial wall. These findings suggest that the injury response is independent of endothelial recovery. Injury leads to loss of endothelial and SMC function transiently but recovery of both occur regardless of hypercholesterolemia. The injured vessels were stiffer and much less compliant than uninjured or injured arteries from C57 mice. Loss of compliance is a common feature of atherosclerotic arteries and contributes to the progression of disease. Arterial stiffness is an independent predictor of adverse cardiovascular events (32) and has been shown to promote the development of atherosclerosis in a primate model (33).

The role of NO in atherosclerosis remains controversial with both deleterious and protective roles for NO and NOS reported (3,5,8,34–37). However, it is well established that NO inhibits IH. We (15,16) and others (17) have reported NOS gene therapy can reduce IH in several normal animal models. In the setting of hypercholesterolemia, the redox environment would reduce the efficacy of NO and yield reactive nitrogen species (8,9). Hayashi et al (38) reported divergent responses to eNOS vs iNOS gene transfer in rabbits. eNOS over-expression improved endothelium dependent vasodilation, reduced plaque size, and decreased nitrotyrosine staining. iNOS gene transfer, however, exacerbated atherogenesis. Using NO donors, Abbasi et al (39) failed to show a reduction in IH and atherosclerosis in a hypercholesterolemic rabbit model. Our current study revealed that iNOS gene transfer inhibited injury induced medial/intimal changes in ApoE KO mice. The difference between our findings and Hayashi et al (38) may reside in the disease model. They created vascular injury to initiate atherosclerosis in rabbits. Gene transfer was performed after lesion formation. The failure of the NO donor (39) may be due to the half-life of the donor. Our findings support the ability of NO to block IH and atheroma formation, lending further enthusiasm for NO based therapies. Despite the dramatic inhibition of neointima/atheroma formation by NO, vasomotor function remained impaired. At the 2 wk time point, injured arteries treated with AdiNOS or AdlacZ were minimally responsive to methacholine, suggesting that endothelial recovery had not been accelerated. However, SMC function at this time point was also impaired. Therefore, it is not possible to determine the effect of NO on endothelial recovery on a functional level. Future studies are needed to determine if the architectural protection of the arterial wall by NO translates into vasomotor preservation.

In summary, hypercholesterolemia impairs endothelium dependent vasomotor function and occurs early. It also enhances the vascular injury response leading to intimal/medial lesions that resemble atheroma and NO therapy can significantly protect against these structural changes. The influences of hypercholesterolemia on endothelial dysfunction and the injury response may involve different pathways with oxidative stress influencing the endothelium and inflammation contributing to injury induced structural changes. These pathways may be regulated by the magnitude of the hypercholesterolemia. Our findings illustrate the adverse consequences of short durations of hypercholesterolemia and support the early correction or prevention of hypercholesterolemia. While few therapies that have made an impact in the setting of atherosclerosis, NO based therapies may prove to be of benefit.

ACKNOWLEGMENTS

This study was supported by R01-HL-57854 (ET), Pacific Vascular Research Foundation Faculty Research Award (ET), and PO1- HD-30367 (RG). We gratefully acknowledge Dr. Simon Watkins, Department of Cell Biology, University of Pittsburgh, for expert assistance in blood vessel imaging.

Footnotes

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DISCLOSURES The authors have nothing to disclose.

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