(Circulation. 1999;100:940-943.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Mononuclear Cell Adherence to Cultured Endothelium Is Enhanced by Hypertension and Insulin Resistance in Healthy Nondiabetic Volunteers
From the Department of Medicine, Stanford University School of Medicine, Stanford, Calif.
Correspondence to Gerald M. Reaven, MD, Shaman Pharmaceuticals, Inc, 213 E Grand Ave, South San Francisco, CA 94080-4812.
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Abstract |
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Background—This study was initiated to compare the adherence to cultured endothelial cells of mononuclear cells isolated from normotensive and hypertensive individuals.
Methods and Results—Mononuclear cell binding to endothelium was greater in patients with hypertension (32±1 versus 25±2; P<0.001) than in normal volunteers. There was a significant relationship (r=0.42, P<0.01) between mononuclear cell binding and mean arterial pressure, independent of differences in age, sex, and body mass index. A significant relationship also existed between insulin resistance (estimated by the steady-state plasma glucose concentration during the insulin suppression test) and mononuclear cell binding in both the normotensive (r=0.86, P<0.001) and hypertensive (r=0.74, P<0.001) groups. Furthermore, multiple regression analysis demonstrated an independent relationship (P<0.001) between mononuclear cell binding and both steady-state plasma glucose and hypertensive status.
Conclusions—These results indicate that both hypertension and insulin resistance lead to changes in mononuclear cells that increase their adherence to cultured endothelial cells.
Key Words: cells • endothelium • hypertension • insulin
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Introduction |
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Although high blood pressure is a well-recognized risk factor1 for coronary heart disease, the link between hypertension and atherogenesis is not fully understood. It has recently been shown that patients with hypertension tend to be insulin resistant, glucose intolerant, hyperinsulinemic, and dyslipidemic, with a high plasma triglyceride and a low HDL cholesterol concentration, recognized risk factors for coronary heart disease.2 3 If these changes account for the increased risk of coronary heart disease in patients with hypertension, the mechanistic link between this syndrome and enhanced atherogenesis is not known. It has been proposed that mononuclear cells play a central role in early atherogenesis by adhering to endothelial cells, migrating through endothelial junctions, and taking up lipid,4 and some experimental evidence suggests that mononuclear cell adhesion to endothelium is increased in atherosclerosis.5 The possibility that similar changes may occur in patients with hypertension is supported by evidence of enhanced monocyte adhesion to endothelium in animal models of hypertension.6 7 8 However, there are no studies of mononuclear cell adhesion to endothelium in patients with essential hypertension. Furthermore, before insulin resistance is common in patients with hypertension and/or hypertriglyceridemia,2 3 9 the possibility that this abnormality might contribute to enhanced mononuclear cell binding seemed worthy of evaluation. This study was initiated to (1) compare adherence to endothelium of mononuclear cells isolated from hypertensive and normotensive patients and (2) evaluate the effect of insulin resistance and its consequences on this measurement.
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Methods |
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The study was performed on 48 volunteers: 22 patients (8 men and 14 women) had high blood pressure and 26 (10 men and 16 women) were healthy volunteers. The majority (71%) of the subjects were of European ancestry, with the remainder being of Asian (25%), African (2%), and Hispanic (2%) ancestry. Blood pressure was measured with a sphygmomanometer, with subjects recumbent for
10 minutes;
diastolic blood pressure was defined as the disappearance
of Korotkoff sounds (phase 5). The hypertensive group consisted of 17
patients taking antihypertensive agents and 5 untreated individuals
with blood pressure >150/90 mm Hg. Seven patients were treated
with ACE inhibitors, singly4 or in combination
with hydrochlorothiazide3 ; 5 with calcium
channel antagonists, singly3 or in combination
with a ß-receptor antagonist; 2 each receiving either an
-receptor or ß-receptor antagonist; and 1 with
hydrochlorothiazide alone. Other than hypertension, individuals enrolled in the study were defined as healthy on the basis of medical history, physical examination, and results of routine laboratory tests and electrocardiography, in addition to being nondiabetic.10 The study protocol was approved by the Stanford University Institutional Review Board, and written, informed consent was obtained from all subjects.
All studies were performed at the General Clinical Research Center of Stanford University Medical Center after an overnight fast. Degree of obesity was estimated by body mass index. Venous blood was used for measurement of plasma cholesterol, triglyceride, and HDL cholesterol concentrations as described previously.11 Plasma glucose12 and insulin13 concentrations were determined before and 30, 60, 90, 120, and 180 minutes after 75 g of oral glucose. The total integrated area of the plasma concentrations during this 180-minute period was used to quantify plasma glucose and insulin responses. Insulin-mediated glucose disposal was estimated by a modification of the insulin suppression test as validated by our laboratory.3 After an overnight fast, an intravenous catheter was placed in each of the patient's arms. Blood was sampled from 1 arm for measurement of plasma glucose and insulin concentrations, and the contralateral arm was used to administer test substances. Somatostatin was infused (250 µg/h in a solution containing 2.5% wt/vol human serum albumin) to suppress endogenous insulin secretion. Simultaneously, insulin and glucose were infused at rates of 25 mU · m-2 · min-1 and 240 mg · m-2 · min-1, respectively. Blood was sampled every 30 minutes until 150 minutes had elapsed and then every 10 minutes for another 30 minutes. The values obtained at 150, 160, 170, and 180 minutes were averaged to represent the steady-state plasma glucose (SSPG) and insulin concentrations achieved during the infusion. Because steady-state plasma insulin concentrations are similar in all individuals, SSPG concentrations provide a direct estimate of insulin-mediated glucose disposal in each individual: the lower the SSPG, the more insulin sensitive the individual.
Blood was drawn after an overnight fast for isolation of mononuclear
cells for adhesion studies.13 14 Viability of isolated
cells was assessed by trypan blue exclusion. Previous results from our
laboratory13 14 have shown that the distribution of
mononuclear cells isolated in this manner is similar in normal
individuals and in patients with
hypercholesterolemia or type 2 diabetes: 
3%
to 10% monocytes, 45% T lymphocytes, and 45% B
lymphocytes.
Binding assays were performed with ECV 304 cells, a human umbilical vein endothelial cell–derived transformed cell line. Endothelial cells were maintained in M199 with 10% FCS and split into 35-mm-diameter wells on 6-well plates 3 days before adhesion assays, and confluence was confirmed before binding studies. Adhesion of mononuclear cells to endothelial cells was assessed by previously established methods.11 14 Freshly isolated mononuclear cell suspensions (3x106/mL final concentration) were added to the wells containing confluent endothelial monolayers. The 6-well plates were transferred to a rocking platform and rocked at room temperature for 30 minutes. Nonadherent cells were removed, and plates were rocked for an additional 5 minutes with fresh binding buffer. Binding buffer was then replaced with HBSS containing 2% glutaraldehyde to fix remaining cells. Adherent cells were quantified by videomicroscopy using a computer-aided image analysis system (Image Analyst, Automatix Corp).
Data are expressed as mean±SEM. Student's nonpaired t test was used to compare groups in terms of their demographic and metabolic covariates. Differences in mononuclear cell binding were compared by 1-way ANOVA before and after adjustment for differences in age, sex, SSPG, and body mass index. Finally, the relationship between mononuclear cell binding and relevant covariates was estimated by Pearson correlation coefficients and multiple regression analysis.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Baseline characteristics of the 2 experimental groups are shown in Table 1
. Even though 17 patients
with hypertension were receiving antihypertensive agents, their mean
arterial blood pressure (MAP) was still significantly
higher (P<0.001). In addition, mononuclear cell adherence
was significantly (P<0.001) increased in cells isolated
from the hypertensive group (32±1 versus 25±2 cells per high-power
field). However, none of the other differences between measured
variables in the 2 groups were significantly different.
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This relationship between MAP and mononuclear cell binding in all
subjects is illustrated in Figure 1 and
indicates that the higher the blood pressure, the greater the
mononuclear cell adherence (r=0.42, P<0.01). The
relationship between MAP and mononuclear cell binding persisted when
adjusted for differences in age, sex, and body mass index.
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), treated
hypertensive (•), and untreated hypertensive (
).
To evaluate the impact on mononuclear cell adherence of potentially
important modulators in addition to hypertension, Pearson correlation
coefficients were calculated between mononuclear cell binding and the
variables listed in Table 1
. These results are shown in
Table 2 and indicate that SSPG, insulin,
and triglyceride concentrations were significantly
correlated with mononuclear cell binding in the entire population.
However, only the relationship between SSPG concentration and
mononuclear cell binding remained statistically significant when the 2
groups were analyzed separately (Table 2 and Figure 2).
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Multiple regression analysis was used to quantify the impact of
mononuclear cell binding on the variables measured. The results in
Table 3 indicate that SSPG was strongly
and independently related to mononuclear cell binding
(P<0.001). The only other variable independently
related to mononuclear cell binding was MAP (P=0.067).
However, it should be remembered that the majority of the patients with
high blood pressure were receiving antihypertensive treatment.
When the diagnosis of hypertension, treated or untreated, replaced
MAP in the model (Table 4), it was also
independently related to mononuclear cell binding
(P<0.001). It should be noted that the relationship between
SSPG and mononuclear cell binding remained highly significant
(P<0.001) when hypertensive status was in the model.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of this study have shown that mononuclear cells isolated from patients with essential hypertension adhere with increased avidity to cultured endothelium compared with mononuclear cells from normotensive individuals. It should be emphasized that this finding obtained even though the blood pressure of 17 patients was lower after treatment with antihypertensive drugs. Thus, it appears that a history of hypertension is at least as important as the actual blood pressure in enhancing mononuclear cell adherence, a conclusion supported by multiple regression analysis showing that hypertension status was related to a greater degree to mononuclear cell binding than was the actual blood pressure. These data provide a possible explanation for the increase in atherosclerotic lesions in the coronary arteries of patients with hypertension described by the International Atherosclerosis Project.15 In addition, the results have demonstrated a highly significant relationship between insulin resistance (and/or hyperinsulinemia) and adherence of mononuclear cells to endothelium. This relationship was seen in the entire population of 48 subjects, as well as when the normotensive and hypertensive groups were analyzed separately. Finally, multiple regression analysis identified either SSPG or fasting insulin concentrations as being related to mononuclear cell binding independently of any other variable measured.
Unfortunately, the present results do not provide insight into the processes responsible at the molecular level for our findings, and there are methodological issues that impair our ability to make this connection. For example, controversy exists as to whether the ECV304 cell line should be used for adhesion molecule research, with evidence both for16 17 and against.18 Furthermore, we did not assess the state of activation of the isolated mononuclear cells, another variable that might contribute to the difference in adherence to the cultured endothelial cells. Finally, the results presented are not necessarily related to the process of atherogenesis. Thus, it could be argued that the mononuclear cell preparations used in this study consist of, at the most, only 10% monocytes. However, mononuclear cells from patients with hypercholesterolemia are hyperadhesive for endothelial cells, and the increase in adhesiveness involved monocytes, T lymphocytes, and to a lesser degree, B lymphocytes,14 a finding of significance in that both monocytes and T lymphocytes constitute major components of the atherosclerotic plaque.4 19
In conclusion, we have presented evidence of a highly significant relationship between insulin resistance and enhanced binding to cultured endothelial cells of mononuclear cells isolated from both normotensive and hypertensive individuals. The ECV304 endothelial cells were used as a tool: to serve as a neutral substrate to see whether there was a difference in the adherence to endothelium of mononuclear cells from insulin-sensitive versus insulin-resistant individuals. Our lack of knowledge about why mononuclear cells from insulin-resistant individuals bound with greater avidity should not negate the fact that we have identified a possible cellular mechanism to explain why insulin-resistant individuals are at increased risk of coronary heart disease.20
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Acknowledgments |
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This study was supported by research grants RR-00070 and HL-08506 and training grant HL-07708 from the National Institutes of Health.
Received May 7, 1999; revision received May 24, 1999; accepted June 2, 1999.
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References |
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|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Kannel WB, Schwartz MJ, McNamara PM. Blood pressure and risk of coronary heart disease: the Framingham study. Dis Chest. 1969;56:43–52.
2.
Fuh MM-T, Shieh S-M, Wu D-A, Chen Y-DI, Reaven GM.
Abnormalities of carbohydrate and lipid metabolism in
patients with hypertension. Arch Intern Med. 1987;147:1035–1038.
3.
Shen D-C, Shieh S-M, Fuh M-T, Wu D-A, Chen Y-DI,
Reaven GM. Resistance to insulin-stimulated glucose uptake in patients
with hypertension. J Clin Endocrinol Metab. 1988;66:580–583.
4. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
5.
Tsao PS, McEnvoy LM, Drexler H, Butcher EC, Cooke JP.
Enhanced endothelial adhesiveness in
hypercholesterolemia is attenuated by
L-arginine. Circulation. 1994;89:2176–2182.
6.
Clozel M, Kuhn H, Hefti F, Baumgartner HR.
Endothelial dysfunction and
subendothelial monocyte macrophages in
hypertension: effect of angiotensin converting enzyme
inhibition. Hypertension. 1991;18:132–141.
7. Tropea BI, Huie P, Cooke JP, Tsao PS, Sibley RK, Zarins CK. Hypertension-enhanced monocyte adhesion in experimental atherosclerosis. J Vasc Surg. 1996;23:596–605.[Medline] [Order article via Infotrieve]
8.
Tsao PS, Niebauer J, Buitrago R, Lin PS, Wang BY,
Cooke JP, Chen YD, Reaven GM. Interaction of diabetes and hypertension
on determinants of endothelial adhesiveness.
Arterioscler Thromb Vasc Biol. 1998;18:947–953.
9. Reaven GM. Role of insulin resistance in human disease. Diabetes. 1988;37:1595–1607.[Abstract]
10. National Data Group. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes. 1979;28:1039–1057.[Medline] [Order article via Infotrieve]
11. Carantoni M, Abbasi F, Chu L, Chen Y-DI, Reaven GM, Tsao PS, Varasteh B, Cooke JP. Adherence of mononuclear cells to endothelium in vitro is increased in patients with NIDDM. Diabetes Care. 1997;20:1462–1465.[Abstract]
12. Kadish AH, Litle RH, Sternberg JC. A new and rapid method for determination of glucose by measurement of rate of oxygen consumption. Clin Chem. 1968;14:116–131.[Abstract]
13. Hales CN, Randle PJ. Immunoassay of insulin with insulin-antibody precipitate. Biochem J.. 1968;88:137–146.
14.
Theilmeier G, Chan JR, Zalpour C, Anderson B, Wange BY,
Wolf A, Tsao PS, Cooke JP. Adhesiveness of mononuclear cells in
hypercholesterolemic humans is normalized by dietary
L-arginine. Arterioscler Thromb Vasc Biol. 1997;17:3557–3564.
15. McGill HC Jr, Arias-Stella J, Carbonell LM, Peylayo Correa MD, DeVeyra EA Jr, Denoso SD, Eggen DA, Galindo L, Guzman MA, Lightenberger E, Joken AC, McGarry PA, McMahan CA, Montenegro MR, Moosy J, Perez-Tamayo R, Restrepo C, Robertson WB, Salas J, Solberg LA, Strong JP, Tejada C, Wainwright J. General findings of the International Atherosclerosis Project. Int Arch Pathol.. 1968;18:498–502.
16. Takahashi K, Sawasaki Y, Hata JI, Mukai, K, Goto, T. Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell Dev Biol.. 1990;25:265–274.
17. Takahashi K, Sawasaki Y. Rare spontaneously transformed human endothelial cell line provides a useful new research tool. In Vitro Cell Dev Biol. 1992;28A:380–382.
18. Stannard AK, Bradley NJ, Owen JS. Evaluation of the ECV304 spontaneously transformed HUVEC cell line for adhesion molecule research. Biochem Soc Trans.. 1997;25:486S.[Medline] [Order article via Infotrieve]
19. Hansson GK, Hom J, Johasson K. Detection of activated T-lymphocytes in human atherosclerotic plaque. Am J Pathol.. 1989;135:169–175.[Abstract]
20.
Yip J, Facchini FS, Reaven GM. Resistance to
insulin-mediated glucose disposal as a predictor of
cardiovascular disease. J Clin Endocrinol
Metab. 1998;83:2773–2776.
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