Background Studies in both animals and humans with raised lipid levels have demonstrated abnormalities in vascular function usually manifested by an impairment in endothelium-dependent vasorelaxation. This is believed to be an early event in atheroma formation. There are few data on the effects on vascular function in humans of lowering serum lipids. We conducted a study to investigate the effects of cholesterol reduction on the in vitro function of human peripheral small arteries in middle-aged patients with hypercholesterolemia.
Methods and Results Subcutaneous gluteal fat biopsies were taken from 18 hypercholesterolemic (HC) patients (mean±SEM serum total cholesterol, 9.7±0.57 mmol/L) and 16 age- and sex-matched control subjects (mean cholesterol, 4.69±0.18 mmol/L). Subcutaneous small arteries (internal diameter, <330 μm) were dissected and mounted on a wire myograph for isometric tension measurements. The HC patients showed impaired relaxation to acetylcholine (10−9 to 10−6 mol/L) after preconstriction with the thromboxane A2 analogue U46619 (10−6 mol/L, mean maximum relaxation, 42.9±5.4%) compared with control subjects (85.7±4.0%, P<.00001). Incubation with the nitric oxide substrate l-arginine (3 mmol/L) improved the endothelium-dependent vasorelaxation response to acetylcholine (70.9±6.0%, P<.01) in patients but not in control subjects. Also, there was a smaller but significant difference in responses to the endothelium-independent agent sodium nitroprusside (10−9 to 10−6 mol/L) between the HC group (mean maximum relaxation, 76.9±6.0%) and the control subjects (89.7±6%; P<.01). A total of 10 patients had a second gluteal skin biopsy and repeat functional studies after successful cholesterol-lowering therapy after a mean period of 9.9±4.7 months. A significant reduction in total and LDL cholesterol was achieved (5.29±0.2 and 3.23±0.21 mmol/L, respectively; P<.001). This restored vasorelaxation to control values in response to both acetylcholine (mean maximum relaxation, 83.3±3.8%; P<.0001) and sodium nitroprusside (87.9±4.8%, P<.01). Although both groups were normotensive, there were significantly higher blood pressures in the HC group compared with control subjects (139±4.1 versus 123±3.0 mm Hg systolic, P<.01; 84±1.3 versus 75±2.2 mm Hg diastolic, P<.01). There was no difference in initial blood pressures between the entire group of 18 and the 10 patients who had repeat biopsies. The blood pressures fell to control values after cholesterol reduction (129.33±4.93 mm Hg systolic and 72.33±2.93 diastolic mm Hg, P<.02 relative to pretreatment values).
Conclusions These results demonstrate abnormalities of both endothelium-dependent and -independent relaxation in human peripheral small arteries that are normalized with effective lipid lowering. The changes in blood pressure may have been secondary to the improvement in vascular function.
The early processes at work in the development of atherogenesis have provoked much interest in the interactions between serum cholesterol and the vascular wall. However, in the small arteries that form the proximal resistance vasculature, atheromatous plaques are not observed.1 In the human heart, recent indirect evidence has been reported that hypercholesterolemia causes impairment of endothelium-dependent dilation in intramyocardial small blood vessels.2 A study of forearm blood flow has also demonstrated blunted responses to both methacholine and nitroprusside in hypercholesterolemia, suggesting that hyperlipidemia can both compromise endothelium-dependent dilatation and have a direct action on smooth muscle function.3 The possibility exists that these phenomena may precede atheroma formation in upstream medium-size and large arteries while representing a widespread abnormality in the peripheral vasculature that persists and interferes with vascular tone and pressure homeostasis. Certainly there is evidence for changes in aortic compliance and endothelial function in young adults and children with hypercholesterolemia4 5 and some evidence that blood pressure is higher in patients with hyperlipidemia.6
It is of further interest that studies of hyperlipidemia in animals suggest that the endothelial dysfunction can be reversed with treatment,7 8 and two studies in humans have reported that reduction of serum cholesterol can improve epicardial coronary artery dilatation9 and coronary blood flow.10 However, there have been no in vitro studies of small arteries from patients with hyperlipidemia either before or after treatment.
This in vitro study adds important data to the in vivo forearm blood flow studies. The technique allows isolated intact small arteries, which are believed to be important as peripheral resistance vessels, to be studied free from confounding influences such as heart rate, varying blood flow, forearm length, and uncertain drug delivery. These isolated vessel techniques offer significant advantages for pharmacological studies.
The purposes of the present study were (1) to examine in vitro the contractile and relaxing properties of small resistance arteries from patients with hypercholesterolemia before and during therapy and (2) to elucidate whether both endothelium-dependent and -independent dilator functions were impaired in hypercholesterolemia, because this is controversial in humans.11 The results indicate that both processes are affected and that treatment can restore their integrity relatively quickly. Moreover, there are interesting effects on systemic arterial blood pressure that may lead to new avenues of research.
Eighteen patients were studied who had a raised total serum cholesterol of >7.5 mmol/L despite 3 months of the standard Step-1 diet. These patients were recruited from the Hospital Lipid Clinic. Care was taken to exclude patients with secondary hyperlipidemia and patients already receiving lipid-lowering drugs, hormone supplements, or antihypertensive therapy. Also, no patients were included who were taking proprietary medications such as vitamins, antioxidants, or fish oils. No patients had familial hypercholesterolemia.
This study population was compared with 16 healthy age- and sex-matched control subjects who were selected on the basis of having a total serum cholesterol of <5.2 mmol/L. These individuals responded to an advertisement placed in a local newspaper.
Patients and control subjects underwent complete clinical examination and had blood drawn for a fasting lipid profile and lipid subfraction analysis. Blood pressure was recorded from the right arm with the use of a standard mercury sphygmomanometer with the participant in the seated position after a 15-minute rest. All patients were seen between 8:30 and 11:00 am, and the mean value of three successive readings was noted.
Small Artery Study
A sample of skin and underlying gluteal fat was taken with the participant under local anesthetic as previously described.12 The specimen was immediately placed into a cold physiological salt solution (PSS) of the following composition (in mmol/L): 119 NaCl, 25 NaH2CO3, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 2.5 CaCl2, 5.5 glucose, and 0.026 EDTA.
Intact small arterial segments 2 mm long were then dissected while under a light microscope and mounted as ring preparations in a wire myograph.12 Vessels were kept at 37°C in PSS gassed with 95% O2–5% CO2 to maintain a constant pH 7.45.
The vessel was set to an internal circumference (Lo) as determined previously to allow isometric tension experiments.13 The circumference that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg (L100) was found with the use of the law of Laplace (ΔP=ΔT/r where ΔP is transmural pressure, ΔT is tension, and r is radius). Lo was then taken as 0.9 L100 and the normalized internal diameter was taken as Lo/Π.
The vessels were stimulated three times (2 minutes) with 5 μmol/L norepinephrine for assessment of tissue viability.
Constrictor function was examined by cumulative dose-response determinations to norepinephrine and the thromboxane analogue U46619 (10−9 to 10−5 mol/L). Vasodilation was assessed by constructing dose-response curves to acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) in increasing concentrations ((10−9 to 10−5 mol/L) after a stable preconstriction with U46619 10−6 mol/L. Vessels were then incubated with the nitric oxide substrate l-arginine (3 mmol/L) for 30 minutes, and the dose-response curve to acetylcholine was repeated.
The hypercholesterolemic patients then began receiving appropriate drug treatment and were followed up on a routine basis at 3-month intervals by physicians in the lipid clinic. When a satisfactory lipid profile had been achieved, a second skin biopsy was taken and the functional studies were repeated. We had no control of the hypolipidemic medication chosen and were interested in cholesterol reduction, not specific drug effects.
All patients and control subjects were fully informed of the nature of the study and gave written consent. The study was approved by the local ethics committee.
Values are given as mean±SEM. ANOVA for repeated measures was used to compare dose-response curves. Student’s t tests were used to compare paired or unpaired data. Linear regression analysis was performed for selected variables. A value of P<.05 was considered to indicate significance.
The groups of patients and control subjects were well matched for age and sex, but the patients had significantly higher levels of total and LDL cholesterol (Table 1⇓). One patient had type III hyperlipidemia, and all other patients had type IIb hyperlipidemia. There was no significant difference in HDL cholesterol or triglyceride levels between the two groups (Table 1⇓). Although blood pressures were well within the normal range, there was a significant difference between systolic and diastolic pressures for the two groups (P<.01). Smoking habits were similar in both groups, as was weight (Table 1⇓).
Pretreatment Small Artery Study
Small artery internal diameters were similar for the two groups (312±10 versus 316±12 μm). There were no significant differences in contractile vascular responses between the two groups to either norepinephrine or U46619 (Figs 1⇓ and 2⇓).
The arteries from hypercholesterolemic patients had markedly impaired endothelium-dependent relaxation compared with responses for vessels from control subjects. The mean maximum relaxation to acetylcholine was 85.7±4.0% in the control group and 49.9±5.4% in the group of hypercholesterolemic patients (Fig 3⇓). In all participants in the small artery study, there were significant correlations of both total serum cholesterol and LDL cholesterol with the degree of endothelial dysfunction (maximum relaxation to acetylcholine) (r=.753, P<.0001; r=.785, P<.0001, respectively). There was no correlation between HDL or LDL-to-HDL ratio and endothelial function. There also was no correlation between nonendothelial function and any lipid fraction.
In arteries from patients with hypercholesterolemia, there was a significant impairment in endothelium-independent relaxation provoked by sodium nitroprusside (Fig 4⇓).
Response to l-Arginine
Preincubation with l-arginine (3 mmol/L) improved acetylcholine-induced endothelium-dependent relaxation in small arteries from hypercholesterolemic subjects (mean maximum relaxation, 70.9±6.0%; P<.01) but did not restore the dysfunction to normal values (P<.02) (Fig 5⇓). l-Arginine had no effect on the responses from vessels from control subjects, and in fact the sensitivity curve was shifted to the right (Fig 5⇓), although this did not attain statistical significance.
Posttreatment Small Artery Study
A total of 10 patients underwent successful treatment according to European Atherosclerosis Society guidelines with significant reduction in both total and LDL cholesterol to 5.29±0.2 and 3.23±0.21 mmol/L, respectively (Table 2⇓⇓) (P<.001), and the patients agreed to undergo a second biopsy to permit the in vitro examination of peripheral small arteries after normalization of their lipid profiles. Two patients refused to undergo a repeat procedure, and 1 patient was lost to follow-up. So far, the remaining patients have failed to respond adequately to treatment, as judged by the treating physician. The drug treatments used are shown in Table 2⇓.
The average time for treatment to normalize lipids was 9.9±4.7 months. Examination of the responses of the preconstricted small arteries to incremental concentrations of acetylcholine demonstrated that the previously impaired endothelium-dependent relaxation was restored to that seen in the control population (Fig 6⇓). Similarly, endothelium-independent relaxation effected by challenging the preconstricted vessels with sodium nitroprusside was completely returned to normal values (Fig 7⇓).
Despite the lack of change in body weight during therapy for the 10 patients reexamined (69.3±2.2 versus 69.5±2.3 kg), it was of interest that mean systolic and diastolic blood pressures both fell significantly (7.7±2.7 and 6.9±2.3 mm Hg, respectively; P<.02). The blood pressures of the control subjects remained the same when taken at a later date (123±5.0 mm Hg systolic and 77±3.6 mm Hg diastolic). There did not appear to be any correlation between the magnitude of cholesterol reduction and the degree of blood pressure drop.
This is the first in vitro examination of segments of small arteries from patients with hypercholesterolemia. It demonstrates that increased serum cholesterol level is associated with a marked impairment in endothelium-dependent dilatation, which is a phenomenon reported using indirect assessments of arterial function such as venous occlusion plethysmography.3 11 The extent of the dysfunction is dependent on the degree of hypercholesterolemia, although the results are relative to a population of middle-aged patients with severe hyperlipidemia and may not be directly extrapolated to other patient groups. However, the present study went further than previous work. First, it suggests that endothelium-independent relaxation is also impaired, a finding reported by some using other techniques but not by others.3 Second, it has demonstrated that, although the abnormality of endothelium-dependent dilatation is improved if arteries are exposed to the nitric oxide substrate l-arginine, it is not restored to normal. Third, restoration of the total serum cholesterol to normal with drug therapy is associated with complete recovery of endothelial integrity. Finally, it was of interest that the blood pressures of patients with hypercholesterolemia were higher than those of matched control subjects, although this was an incidental finding and was not the major parameter being examined. The blood pressure decreased when the increased cholesterol improved, which raises interesting questions about further study. Although this theoretically could be explained by improved vascular tone, this part of the study was uncontrolled and therefore we cannot speculate regarding its clinical relevance.
The mechanism by which an abnormal lipid profile impairs arterial function has been the subject of considerable attention. It is important to bear in mind that, although endothelium-dependent dilatation is most affected, endothelium-independent relaxation is also influenced, albeit to a lesser extent. With regard to endothelium-dependent relaxation, it appears that there must be a reduced bioavailability of nitric oxide. This is synthesized from the substrate l-arginine,14 and it has been demonstrated that an infusion of l-arginine can restore endothelium-dependent relaxation to normal levels in hypercholesterolemic rabbits.15 Furthermore, similar findings have been reported in the human coronary circulation16 and the forearm vasculature.17 However, in the present study, preincubation in vitro with l-arginine improved endothelial integrity slightly but did not restore normal function. In addition, it has been observed that hypercholesterolemia produces an increase in the release of nitrosylated compounds from the rabbit aorta.18 Therefore, it appears that there is evidence that nitric oxide synthesis is enhanced in hypercholesterolemia but that its degradation is faster or it is released in a less bioactive form. Ultimately, the vasodilatation brought about by these endothelial factors occurs via the same mechanism activated by endothelium-independent agonists such as sodium nitroprusside, ie, by stimulation of guanylate cyclase within vascular myocytes and formation of a nitrosyl-porphyrin complex.19 20 The activity of guanylate cyclase may be affected directly by hypercholesterolemia as a result of a change in the redox state of the vascular smooth muscle.21 Therefore, there is an emerging body of evidence to suggest that increased serum cholesterol levels can interfere with vascular dilator mechanisms at a variety of points.22 In further study, we would like to examine vasodilators that do not act through nitric oxide release.
The exact lipid fraction that is responsible for these findings also is uncertain. It seems clear that triglycerides are not contributing, as we have shown little change in triglyceride profiles after treatment yet demonstrated good functional improvement. Among the cholesterol subfractions, the most likely candidate is the oxidized LDL fraction. Recently, it has been reported that oxidized LDL could attenuate endothelium-dependent relaxation in vitro.23 24 This impairment could be overcome by HDL. Zeiher et al25 have also shown that elevated HDL cholesterol levels ameliorate abnormal vasoconstriction in human coronary arteries. High levels of oxidized LDL may generate free radical species from endothelial cells,26 and it is known that superoxide anions in particular are released from vessels from hypercholesterolemic animals and that they can inactivate nitric oxide.27 This theory is supported by the work of Raij et al,28 which has demonstrated that hypercholesterolemia promoted endothelial dysfunction in vitamin E–deficient (a potent antioxidant) and selenium-deficient rats, and this work provides further evidence for an attack on guanylate cyclase activity being central to the mechanism underlying this phenomenon.
Treatment of hypercholesterolemia with drug therapy restored the lipid profile of 10 of the study population to normal levels within months, and this was associated with improved endothelium-dependent and -independent dilator function and a decrease in systemic arterial blood pressure. Given the above evidence, the most likely explanation is that a reduction in available LDL cholesterol lowers its oxidation potential and generates free radical species. In this regard, there is increasing evidence for the protective effects of antioxidants such as vitamin E in heart disease29 30 and in vitro studies.28 31 Certainly, these dramatic improvements in arterial function have been accomplished within a time frame much shorter than that required to observe the regression of atheromatous plaques and suggest that more wide-ranging improvements in circulatory integrity are possible with control of an abnormal lipid profile.
In summary, in vitro studies of small arteries from patients with hypercholesterolemia demonstrate abnormalities of both endothelium-dependent and -independent relaxation but normal vascular sensitivity to vasoconstrictor agents. Therefore, it is possible that the deficit is one of guanylate cyclase activation, although we have provided no conclusive evidence for this here. Normalizing the lipids restores arterial function to normal relatively rapidly, and this may explain the fall in blood pressure recorded. In any event, the improvement in vascular function may be important from the clinical perspective of blood pressure homeostasis, and we suggest that studies be designed to look into possible blood pressure–lowering effects of hypolipidemic agents.
The study was funded by grants from the Wolfson Foundation and the British Heart Foundation. The authors thank Tracy Bent for help in the preparation of the manuscript.
- Received November 1, 1994.
- Accepted December 20, 1994.
- Copyright © 1995 by American Heart Association
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