Relationship Between Endothelial Function and Fibrinolysis in Early Hypertension
Abnormalities in fibrinolysis, endothelial function, and glucose and lipid metabolism have been reported in hypertension. This study was conducted to examine the interrelationships between fibrinolytic factors, glucose and lipid metabolism, and endothelial function in hypertension. The effects of administering an angiotensin converting enzyme inhibitor, benazepril, were also examined. Blood levels of the following substances were measured in patients with borderline and mild hypertension (n=50, 51±19 years) and in age-matched controls (n=10): total cholesterol, triglycerides, tissue plasminogen activator activity and antigen, and plasminogen activator inhibitor type 1 activity and antigen. Insulin sensitivity was assessed by oral glucose tolerance test, and endothelial function was assessed by evaluating changes in diameter of the brachial artery during reactive hyperemia as observed by ultrasonography. Activities of tissue plasminogen activator and plasminogen activator inhibitor type 1 were both elevated in the hypertensive patients. Stepwise multiple regression analysis showed that plasminogen activator inhibitor type 1 antigen correlated with insulin sensitivity, total cholesterol levels, and triglycerides levels (P<.01). Endothelial function was negatively correlated with tissue plasminogen activator activity and antigen (P<.01). The chronic administration of benazepril (5–10 mg/d) for 20 weeks improved insulin sensitivity, endothelial function (6.6+3.4→9.0+2.5%, P<.01), and tissue plasminogen activator activity and antigen. These results indicate that abnormalities in fibrinolysis are associated with endothelial dysfunction as well as disorders of glucose and lipid metabolism in patients with borderline and mild hypertension. The treatment of such patients with benazepril appeared to improve the impairment in fibrinolysis and endothelial dysfunction.
- insulin resistance
- tissue plasminogen activator
- plasminogen activator inhibitor type 1
- angiotensin converting enzyme inhibitor
- ACE = Angiotensin converting enzyme
- FDP = fibrinogen/fibrin degradation products
- PAI-1 act = plasminogen activator inhibitor type 1 activity
- PAI-1 ang = plasminogen activator inhibitor type 1 antigen
- PAI-1/tPA = ratio of tPA ang to PAI-1 ang
- Σ BS = sigma plasma glucose
- Σ INS = sigma plasma insulin
- tPA act = tissue plasminogen activator activity
- tPA ang = tissue plasminogen activator antigen
An impairment of fibrinolysis is an established risk factor for such thrombotic events as stroke or myocardial infarction.1–3⇓⇓ Thrombosis and fibrinolysis are interrelated in the development of atherosclerosis.4 Thus, the evaluation of fibrinolysis in the pathophysiology of cardiovascular diseases is essential. In hypertension, a close relationship has been demonstrated between disorders of glucose and lipid metabolism and disturbances of fibrinolysis.5,6⇓ Fibrinolytic activity is primarily determined by the balance between the levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1), both of which are synthesized in the vascular endothelium,7,8⇓ and vascular endothelial injury induces an imbalance in fibrinolysis.4,7,8⇓⇓ Because hypertension and disorders of glucose and lipid metabolism are accompanied by endothelial dysfunction,9 the relationship between endothelial function and fibrinolytic balance needs to be examined in detail and more precisely. However, the relationship between endothelial dysfunction and fibrinolytic imbalance has not yet been thoroughly investigated in the clinical setting.
Some antihypertensive drugs exhibit a beneficial effect on impaired fibrinolysis.10,11⇓ Angiotensin converting enzyme (ACE) inhibitors are reported to have such effects on fibrinolysis,11 as well as on glucose metabolism12,13⇓ and endothelial function.14,15⇓ However, the interactions between the changes in fibrinolysis, glucose metabolism, and endothelial function following treatment with an ACE inhibitor have not been studied.
Recently, a noninvasive means for the clinical evaluation of vascular endothelial function has become available through high resolution ultrasonography.16,17⇓ The present study examined the interrelationships between endothelial function as determined by ultrasonography, fibrinolysis, and glucose and lipid metabolism in patients with borderline or mild hypertension. The effects of long-term treatment with an ACE inhibitor on these epiphenomena were also examined.
We selected consecutively for the present study 50 ambulatory Japanese patients with borderline-to-mild hypertension (24 male, 26 female; mean age, 51±9 years) who visited Teikyo University School of Medicine, Ichihara Hospital, from April 1996 to February 1997. Inclusion criteria were as follows: the subjects had no history of receiving antihypertensive medications; had acceptable ultrasonographic recordings of the heart, carotid artery, and brachial artery; and had average blood pressure levels in systole between 140 and 180 mm Hg, and in diastole between 90 and 105 mm Hg over three consecutive visits prior to the study. Patients with secondary hypertension or other serious medical problem that required specific treatment were excluded from the study. As control subjects, age-matched healthy volunteers (6 males and 4 females; mean age, 46±6 years) were recruited for the determination of brachial endothelial function and its reproducibility, fibrinolytic parameters, and ultra-sonographic examinations of the heart and carotid artery.
Informed consent was obtained from all subjects based on a protocol approved by the Ethics Committee of Teikyo University School of Medicine, Ichihara Hospital. Each patient underwent echocardiography, ultrasonographic examination of the carotid artery, study of brachial endothelial function, oral glucose tolerance testing, and routine laboratory examinations of serum and urine obtained in the fasting state. Patients with mean systolic pressure of <160 mm Hg and diastolic pressure <96 mm Hg during three consecutive visits had educational counseling on modification of lifestyle, and were scheduled to be followed-up without antihypertensive medication at 3-month intervals. Patients with a mean systolic pressure ≥160 mm Hg and/or diastolic pressure ≥96 mm Hg were prescribed an ACE inhibitor, benazepril (5 mg/d) (Novartis) and were followed at the outpatient clinic. If the medication had not reduced systolic pressure to <150 mm Hg and diastolic pressure to <90 mm Hg after 2 weeks of treatment, the dose was doubled. If the systolic pressure remained ≥150 mm Hg or diastolic pressures remained ≥90 mm Hg after 5 weeks of the treatment, amlodipine (5 mg/d) was added to the regimen. If systolic pressure remained ≥150 mm Hg or diastolic pressure remained ≥90 mm Hg at the beginning of the 10th week, the subject was excluded from the study. Thereafter, patients were followed at 4-week intervals at the outpatient clinic. After the systolic pressure had remained below 150 mm Hg and diastolic pressure had remained below 90 mm Hg for 20 weeks, we again performed echocardiography, ultrasonographic examination of the carotid artery, a study of brachial endothelial function, oral glucose tolerance testing, and routine examinations of serum and urine obtained in the fasting state, except for the oral administration of the antihypertensive medication.
Blood Pressure Measurements
Blood pressure was measured in an office setting by the conventional cuff method with a mercury manometer. Patients remained seated for at least 10 minutes before blood pressure measured. Diastolic pressure was determined at the 5th Korotkoff sound.
Ultrasonographic Examinations of the Heart and of the Carotid Artery
The ultrasonographic examinations were performed by the same well-trained sonographer (Y.K.). With the patients in the left semilateral decubitus position, M-mode echocardiograms guided by two-dimensional echocardiography were obtained by a Sonolayer (SSH-160A, Toshiba Co) with a 2.5-MHz or a 3.75-MHz transducer. Data were printed on a split-chart recorder at a recording speed of 50 mm/s. End-diastolic interventricular septal thickness, left ventricular internal diameter, and posterior wall thickness in diastole were measured according to the method based on recommendations of The American Society of Echocardiography and Penn Convention.18 Each dimension was defined as the mean of two M-mode measurements obtained by two different investigators (H.T. and Y.K.). Left ventricular mass was calculated by Devereux’s method.19 The left ventricular mass index was derived from left ventricular mass devided by the body surface area.
After the completion of echocardiographic examination, the patients was placed in the supine position with the neck slightly extended, and the common carotid artery, carotid sinus, and extracranial internal and external carotid arteries were examined (long-axis and short-axis views) with an ultrasonographic system (SSH-160A, Toshiba Co) equipped with a 7.5-MHz transducer. The common carotid artery was examined in a two-dimensional, long-axis view approximately 1 cm upstream from the carotid bifurcation. Thereafter, the transducer was rotated 90° and M-mode tracings of this area of the common carotid artery were obtained in a two-dimensional short-axis view along with simultaneous electrocardiographic recordings on a split-chart recorder (recording speed of 50 mm/s). The presence of an atherosclerotic plaque was defined as a 50% thickening of the wall relative to the surrounding wall. If plaque was observed in a searching area, the transducer was moved to upstream in the common carotid artery. End-diastolic far-wall thickness and end-diastolic and peak systolic internal dimensions were measured using a mean of M-mode images obtained from the right and the left common carotid artery for consecutive three cardiac cycles.
All of the measurements of ultrasonographic examinations used were a mean of two readers (H.T. and Y.K.). The reproducibility of the echocardiogram and the ultrasonographic measurements of the carotid artery in our institute has been previously reported elsewhere.20
Brachial Endothelial Function Test
The brachial endothelial function test, a modified version of Celermajer’s method16 was performed in the morning fasting state by a well-trained cardiologist (H.T.). Images of the brachial artery of the dominant arm in each subject were obtained with an ultrasonographic system (SSH-160A, Toshiba Co) equipped with a 7.5-MHz transducer. A long-axis view of the straight segment (at least 1 cm) of the brachial artery was obtained immediately above the antecubital fossa. This straight segment was placed at the center of the long-axis image and the diameter of the brachial artery was determined. The center of that image was marked by placing a piece of tape on the skin of the antecubital fossa, and the transducer was rotated 90°. The diameter of the brachial artery in the short-axis view was re-confirmed. Next, M-mode tracings of this area of the brachial artery were obtained in a two-dimensional short axis view together with simultaneous electrocardiographic recordings on a split-chart. After the basal recordings of brachial artery were obtained, reactive hyperemia was produced by inflating the cuff on the upper arm to 20 mm Hg above systolic blood pressure for 3 minutes. Thirty seconds after the release of the cuff, M-mode tracings of the same area of the brachial artery were obtained. Ten minutes after the release of the cuff, a glyceryl trinitrate spray (0.4 mg) was administered sublingually, and, 90 seconds later, M-mode tracings were obtained of the same area of the brachial artery. The diameter of the brachial artery was determined at the end of the T wave using the mean of M-mode images over three consecutive cardiac cycles. The percentage of changes in diameter of the brachial artery in response to reactive hyperemia or to the administration of glyceryl trinitrate were calculated.
The internal diameter of each brachial artery was measured by two observers (H.T. and Y.K.). Interobserver and intraobserver variability in repeated measurements of identical recordings and in the reproducibility of hyperemia and in the response to glyceryl trinitrate were assessed by an independent study of 10 healthy volunteers and another 6 patients with ischemic heart disease (4 males and 2 females; mean age, 56±9 years). Interobserver and intraobserver variability for repeated measurements of same recordings were 0.040±0.05 mm and 0.020±0.03 mm, respectively. To assess the reproducibility of the hyperemia and the response to glyceryl trinitrate administration, the brachial endothelial function test was performed in healthy volunteers on two occasions at least 2 weeks apart. The mean differences in the percentage of changes in diameter of the brachial artery in response to hyperemia and glyceryl trinitrate administration were 1.3±1.3% and 1.5±1.8%, respectively.
Oral Glucose Tolerance Test
The oral glucose test was performed in the morning after a 12-hour overnight fast. Blood samples were drawn before ingestion and 30 minutes, 60 minutes, and 120 minutes after ingestion of a solution containing 75 g of glucose. Total area under curve of the plasma glucose-time plot (sigma plasma glucose or Σ BS) or insulin-time plot (sigma plasma insulin or Σ INS) were calculated. Insulin sensitivity was defined as sigma plasma glucose/sigma plasma insulin.
Measurements of fibrinolytic and hemostatic variables and lipids were performed on the day of the oral glucose tolerance test on blood samples obtained before the ingestion of glucose (8:30 am). Blood samples for determination of fasting lipid levels were collected into tubes containing EDTA. Plasma triglycerides, total cholesterol and high-density lipoprotein cholesterol levels were measured enzymatically with a Hitachi 731 Analyzer. Blood samples for determination of fibrinolytic parameters were collected with citrate buffer (pH 4.5). The tPA and PAI-1 activities were assessed with a chromogenic assay kit (Spectrolyse tPA/PAI-1 activity assay kit, American Diagnostica). Plasma tPA and PAI-1 antigen levels were measured by enzyme-linked immunosorbent assay (Imulize tPA kit, Biopool AB, PAI-1 ELISA kit, Monozyme). Plasma fibrinogen levels were determined with a one-stage clotting assay kit (Thrombocheck Fib, International Reagents Corp). D-dimer was measured by an enzyme-linked immunosorbent assay (D-Di test, Boehringer Mannheim). Fibrinogen/fibrin degradation products (FDP) was determined by a latex photometric immunoassay method (COBAS MIRA).
Data are expressed as the mean±SD. Statistical analysis was performed using software from SPSS Inc. The significance of differences between the hypertensive patients and control subjects was evaluated by the Student’s unpaired t-test. The paired t-test was employed to evaluate changes subsequent to ACE inhibitor treatment. Linear regression analysis was used to evaluate the inter-correlations among the fibrinolytic and hemostatic parameters and other parameters. Thereafter, stepwise multiple regression analysis was performed on the fibrinolytic and hemostatic parameters and other parameters to identify significant correlations as determined by the linear regression analysis. A level of P<.05 was accepted as statistically significant.
Of the initial 50 patients, 20 patients exhibited borderline hypertension, and 30 patients had mild hypertension on whom initial treatment with benazepril was instituted. Of these 30 patients, 17 patients continued to receive benazepril alone, 6 received benazepril plus amlodipine, and 7 patients were excluded from further study because of cough (n=4), poor control of blood pressure (n=1), or a personal reason (n=2). In addition to the expected differences in blood pressure, left ventricular mass index, tPA activity and antigen, and PAI-1 activity and antigen were higher in the hypertensives than in controls (Table 1). The changes in brachial artery diameter in response to reactive hyperemia and glyceryl trinitrate administration were smaller in the hypertensive patients than in the controls.
Cross-correlations between fibrinolytic and hemostatic parameters and other targeted parameters are presented in Table 2. Linear regression analysis revealed a significant negative correlation between tPA activity and the change in brachial artery diameter in response to either reactive hyperemia or glyceryl trinitrate, while tPA antigen levels revealed a positive correlation with triglycerides levels and a negative correlation with the change of brachial artery diameter in response either to reactive hyperemia (Fig 1) or glyceryl trinitrate. There was a positive correlation between PAI-1 activity and triglycerides levels. In addition, PAI-1 antigen levels displayed correlations with total cholesterol levels, triglycerides levels, Σ INS, and insulin sensitivity. Lastly, fibrinogen correlated significantly with age. The results of stepwise multiple regression analysis (Table 3) revealed several equation variables for these parameters. In addition, by linear regression analysis, the change of brachial artery diameter in response to reactive hyperemia had a negative correlation with sigma plasma glucose (r=−0.29, P<.05), but not with plasma triglycerides, total cholesterol, or high-density lipoprotein cholesterol levels. After the treatment with benazepril alone or benazepril plus amlodipine for 20 weeks, blood pressure and the left ventricular mass index decreased significantly (Table 4). tPA antigen and activity, PAI-1 activity, sigma plasma glucose, insulin sensitivity, and the change in brachial artery diameter in response to reactive hyperemia improved significantly either in patients treated with benazepril alone or benazepril plus amlodipine.
Numerous pathophysiological factors are associated with hypertension,21 and some of these factors, such as the increase in shear stress caused by elevated blood pressure,22 disorders of glucose and lipid metabolism,5,6⇓ atherosclerosis,23 and endothelial cell injury,24 are associated with imparied fibrinolysis. However, previous studies evaluating abnormal fibrinolysis in hypertension have focused only on disorders of glucose and lipid metabolism.5,6⇓ The present study evaluated the relationships between the severity of hypertension, disorders of glucose and lipid metabolism, the severity of atherosclerosis, and the extent of vascular endothelial dysfunction. Although the severity of hypertension was evaluated by both blood pressure values and left ventricular mass index, and the severity of atherosclerosis was evaluated by the intimal-media thickness of the carotid artery, these indices did not correlate with the severity of abnormalities of fibrinolysis. However, parameters of glucose and lipid metabolism were correlated with PAI-1 activity or its antigen levels, while endothelial function was negatively correlated with tPA activity and antigen levels. Thus, abnormalities in fibrinolysis in patients with borderline and mild hypertension are closely associated with disorders of glucose and lipid metabolism and endothelial dysfunction, rather than with the severity of the hypertension or atherosclerosis. Therefore, the results of the present study particularly suggest that PAI-1 activity and antigen levels are affected by disorders of glucose and lipid metabolism, and that tPA activity and antigen levels are affected by endothelial dysfunction. Furthermore, the fact that endothelial function demonstrated only a partial correlation with Σ BS suggests that endothelial function directly affects the fibrinolytic balance.
While normal vascular endothelium is in a nonthrombotic state, disruption of the vascular endothelium results in the synthesis and release of procoagulant and anticoagulant substances from the disrupted vascular endothelium.4,7⇓ Some studies suggest that an increase in tPA antigen level indicates endothelial dysfunction.3,25⇓ The present results are consistent with this hypothesis and confirm that increases in tPA activity and antigen levels reflect endothelial dysfunction in patients with borderline and mild hypertension. The fact that ultrasound examination of the carotid artery in our study did not detect regions of advanced plaque indicates that atherosclerosis was not marked in the present subjects. Although a downregulation of tPA production has been observed in plaque lesions of the aorta,24 the present results suggest that tPA synthesis in the endothelium is activated in the early stages of atherosclerosis.
Beneficial effects of ACE inhibitors on insulin resistance12,13⇓ and impaired fibrinolysis11,26⇓ have been reported, and some studies have indicated that such drugs may improve vascular endothelial function.14,15⇓ Clinically, endothelial function is usually assessed by the blood flow response to acetylcholine or the development of reactive hyperemia.27,28⇓ Both methods induce vasodilation, the former being mediated primarily by nitric oxide and the latter by nitric oxide plus vasoactive substances such as prostaglandins, adenosine, and potassium.29 Some studies have indicated that ACE inhibitors do not affect endothelial function as evaluated by the response to acetylcholine.27,30⇓ However, consistent with the results of the present study, Iwatsubo et al reported that an ACE inhibitor does improve endothelial function as evaluated by the response to reactive hyperemia.15
Angiotensin II increases the synthesis of PAI-1 in the vascular endothelium, and the reduction of angiotensin II levels by an ACE inhibitor improves fibrinolytic balance.31 In addition, insulin also increases PAI-1 synthesis,32 and the improvement in glucose metabolism following treatment with benazepril may have beneficial effects on fibrinolysis. In the present study, benazepril reduced the activity of both tPA and PAI-1 and tPA antigen without altering the ratio of levels of tPA antigen to PAI-1 antigen, thought to be a better index of overall fibrinolytic potential.11,31⇓ Benazepril did not affect plasma levels of FDP or D-dimer. These data suggest that the improvement of endothelial function following benazepril treatment may have a favorable effect on the prevention of thrombogenesis. The results also indicate that assessment of the endothelial function should be considered in the management of patients with hypertension.
The present study demonstrated beneficial effects of benazepril on fibrinolytic abnormalities and endothelial function. However, the numbers of patients were too small to examine whether the coadministration of amlodipine alter those beneficial effects of benazepril. Various vasoactive substances that are synthesized in the vascular endothelium affect hemostasis and fibrinolysis.4,7,8⇓⇓ Further study is necessary to identify the factors that contribute to the abnormal fibrinolysis observed in patients with hypertension. The results of the present study indicate that the fibrinolytic imbalance and endothelial dysfunction are reversible in patients in the early stages of hypertension. The relationship between the fibrinolytic imbalance and endothelial dysfunction and the reversibility of such abnormalities should be examined in patients in the advanced stages of hypertension.
In conclusion, the present study showed that abnormalities in fibrinolysis were associated with disorders of glucose and lipid metabolism, as well as with endothelial dysfunction in patients with borderline or mild hypertension. The treatment of hypertensive patients with an ACE inhibitor appeared to be effective against these abnormalities.
- Received September 16, 1997.
- Revision received October 16, 1997.
- Accepted October 29, 1997.
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