Endometrial angiogenesis throughout the human menstrual cycle.

BACKGROUND: The timing and mechanisms of new blood vessel formation in the endometrium during the menstrual cycle are still largely unknown. In the present study we used the chick embryo chorioallantoic membrane (CAM) as an in-vivo assay for angiogenesis to assess the angiogenic potential of endometrium obtained at different stages of the menstrual cycle. METHODS: Endometrial fragments were explanted onto the CAM and, after 4 days of incubation, slides of the treated area were taken in ovo through a microscope for computerized image analysis. The vascular density index (VDI), a stereological estimate of vessel number and length, was obtained by counting the intersections of vessels with five concentric circles of a circular grid superimposed on the computerized image. RESULTS: We demonstrated that human endometrium has angiogenic potential throughout the menstrual cycle. Furthermore, there was a significant difference in angiogenic response between the stages of the menstrual cycle (P = 0.01). The VDIs of the early proliferative, early and late secretory stage were significantly higher than the VDI of the late proliferative phase. CONCLUSIONS: Elongation of existing vessels during the early proliferative phase as well as growth and coiling of the spiral vessels during the secretory phase may demand far higher angiogenic activity than outgrowth and maintenance of vessels during the late proliferative phase.

The chick embryo chorioallantoic membrane as a model to investigate the angiogenic properties of human endometrium.

The chick embryo chorioallantoic membrane (CAM) is an established in vivo angiogenesis assay. The aim of our study was to assess the angiogenic properties of endometrium and to quantitate the vascular response in an accurate way. Samples of proliferative endometrium (n = 17) and control mouse skin tissue (n = 8) were explanted onto the CAM at day 10 of incubation. Additional controls consisted of normal unmanipulated CAM (n = 12). Four days after grafting, photographs of the explant and the surrounding area were taken in ovo to measure the vascular density index (VDI). The VDI is a stereological estimate of vessel number and length, which was obtained by counting the intersections of vessels with a circular grid superimposed on a computerized image. Endometrium caused a significant increase in VDI as compared to both unmanipulated CAM (p < 0.001) and skin tissue as a control (p < 0.007). The intra-observer variability was 5.2%. This study demonstrates that the CAM assay is a suitable model to assess the angiogenic properties of endometrium. Furthermore, it allows detailed quantitation of the vascular response in an objective and reproducible way. Our findings suggest the CAM to be a promising model to study the role of angiogenesis in both normal human endometrium and diseases involving the endometrium.

Cardiac function in a rat model of chronic aortic stiffness.

Cardiac function was investigated in conscious normotensive rats in which increased aortic stiffness was produced as a result of vascular calcium overload after treatment with vitamin D3 plus nicotine (VDN rats, n = 16; controls, n = 17). Baseline stroke volume, cardiac output, and cardiac response to a venous volume overload were unchanged after 1 mo of exposure to increased aortic stiffness, as were baseline venous return and total vascular capacitance. The latter was estimated from the change in mean circulatory filling pressure after modification of circulatory volume. Cardiovascular reflexes were modified in VDN rats. Bradycardia evoked by an increase in arterial PCO2 (PaCO2) or hypotensive hemorrhage was more pronounced. The PaCO2-induced bradycardia was accompanied by a fall in cardiac output in VDN rats but not in controls. In VDN rats, the attenuation of sympathetic reflexes may explain the slower recovery of blood pressure after hypotensive hemorrhage. In conclusion, a chronic increase in aortic stiffness does not compromise cardiac performance, but cardiovascular reflexes are impaired in VDN rats. Whether this is because of the increase in aortic stiffness or the effect of VDN treatment on the baroreceptors or other components of the reflex arc remains to be elucidated.

A technique to assess aortic distensibility and compliance in anesthetized and awake rats.

A noninvasive ultrasonic technique, based on tracking arterial wall displacements with a vessel wall-tracking device attached to a conventional B-mode imager, to assess end-diastolic aortic diameter (d) and aortic diameter changes during the cardiac cycle (delta d) in anesthetized and awake rats is presented. From these parameters and invasively measured aortic pulse pressure (delta P), aortic distensibility and compliance, the relative and absolute increases in lumen cross-sectional area for a given increase in delta P, respectively, can be calculated. d, delta d, and delta P could be determined with good intra-session (variations per day) and inter-session (variations between days) coefficients of variation (CV). The CVs for delta d were smaller in awake (4.6-6.0%) than in anesthetized rats (7.9-11.0%), probably due to variations in delta P during anesthesia (CV: 9.0-12.3%). The CVs for d in awake (3.3-6.5%) and anesthetized rats (2.6-5.0%) were comparable. In awake rats the CV for delta d, but not for d, increased after implantation of the aortic catheter. It is concluded that d, delta d, and delta P of the aorta can be reliably measured noninvasively in anesthetized and awake rats, allowing the in vivo assessment of aortic distensibility and compliance. The technique is sensitive enough to detect effects of agents on aortic wall properties.

The influence of perindopril and the diuretic combination amiloride+hydrochlorothiazide on the vessel wall properties of large arteries in hypertensive patients.

OBJECTIVE: To compare the cardiovascular effects of 6 months of treatment with the angiotensin converting enzyme inhibitor perindopril and with the diuretic combination amiloride+hydrochlorothiazide, and to study possible persistence of observed treatment effects after discontinuation of antihypertensive therapy. DESIGN: A placebo run-in period preceded a 6-month active-treatment phase in 41 patients with essential hypertension, according to a double-blind, randomized, parallel-group design. Patients received either 4 mg perindopril or 2.5/25 mg amiloride+hydrochlorothiazide once a day. Patients were then studied for a 3-month single-blind placebo run-out period. RESULTS: After 6 months of treatment, systolic blood pressure was reduced significantly by perindopril (supine by 11%, sitting by 10%) and by amiloride+hydrochlorothiazide (supine by 8%, sitting by 12%). Diastolic blood pressure was also decreased significantly by perindopril (supine by 8%, sitting by 11%) and by amiloride+hydrochlorothiazide (supine by 4%, sitting by 9%). Mean arterial pressure decreased significantly during treatment with perindopril (by 9%) and with amiloride+hydrochlorothiazide (by 6%). Cardiac index increased with perindopril (by 6%), because of an increased stroke index (by 5%), but amiloride+hydrochlorothiazide did not change cardiac function. Systemic vascular resistance index decreased significantly more with perindopril (by 14%) than with amiloride+hydrochlorothiazide (by 8%). The distensibility of the common carotid artery was significantly enhanced by perindopril (by 16%), but not changed by amiloride+hydrochlorothiazide (1% difference). The difference between perindopril and amiloride+hydrochlorothiazide for carotid distensibility was statistically significant. The compliance of the common carotid artery tended to be increased more by perindopril (by 7%) than by amiloride+hydrochlorothiazide, which induced a 5% decrease in carotid compliance. After withdrawal of therapy, for both drugs, all treatment-induced changes were reversed to pretreatment values within 7 weeks. CONCLUSION: The distensibility of the elastic common carotid artery was increased by perindopril, but not by amiloride+hydrochlorothiazide. Large-artery properties of the muscular arteries and systemic vascular resistance improved with both drugs, but in general the changes were more pronounced with perindopril than with amiloride+hydrochlorothiazide. The present results indicate a more pronounced effect of perindopril at both macro- and microcirculatory levels, which will consequently lead to a larger decrease in cardiac afterload. After discontinuation of therapy all parameters returned to baseline values within 7 weeks.

Mechanical and contractile properties of in situ localized mesenteric arteries in normotensive and spontaneously hypertensive rats.

An in situ model was developed for studying mechanical properties of mesenteric arteries in rats. A branch of the mesenteric artery was exposed and dissected in normotensive (WKY) and spontaneously hypertensive rats (SHR). A catheter was introduced into the larger branch of the mesenteric artery and connected to a pressure chamber. The artery was submitted to transmural pressures ranging from 0 to 200 mmHg per steps of 25 mmHg and observed using a microscope-video-camera system. The diameter-pressure relations were established under basal conditions, under contraction (phenylephrine 10(-6) M), and after abolition of the smooth muscle tone by potassium cyanide (KCN, 0.1 mg/mL). The arterial segment was then fixed (glutaraldehyde 2.5%), and the wall cross-sectional areas were measured in transverse sections. Compliances, distensibility, wall tensions, and wall stresses were calculated from diameter, pressure, and media thickness values under three conditions. Active tension and active stress were defined as differences in wall stresses and wall tensions calculated under passive and active conditions. Comparison of WKY and SHR when arteries were studied at the respective operating pressure indicates (1) thicker and stiffer mesenteric arteries in SHRs than in WKY rats, (2) similar wall stresses in mesenteric arteries from WKY and SHRs despite larger wall tensions in the hypertensive group, and (3) larger contractility to phenylephrine in SHRs than in WKY mesenteric arteries.

Acute and subacute effects of nicorandil and isosorbide dinitrate on vessel wall properties of large arteries and hemodynamics in healthy volunteers.

Nicorandil (N) and isosorbide dinitrate (ISDN) are vasodilator drugs used in patients with angina. In 24 healthy male volunteers (18-32 years), the acute effect of a single oral dose (20 mg) of N and ISDN on arterial diameter (D), distensibility, and compliance of the elastic common carotid artery (CCA) and the muscular femoral (FA) and brachial (BA) arteries were investigated. The effects on systolic and diastolic blood pressure (SBP, DBP), heart rate (HR), cardiac index (CI), systemic vascular resistance index (SVRI), and venous hemodynamics were also assessed. In addition, the subacute effects after 8 days of treatment with N (2 x 20 mg/day) and ISDN (3 x 20 mg/day) on these parameters were evaluated. After a 20 mg single oral dose, blood pressure decreased significantly more with ISDN (SBP: 6%; DBP: 14%) than with N (SBP: 2%; DBP: 6%), but after 8 days this decrease in blood pressure was not statistically different between ISDN and N. The diameter of CCA increased more with ISDN (11%) than N (5%) acutely as well as subacutely (ISDN: 12%; N: 9%). Heart rate increased only with ISDN (7% acutely, 3% subacutely). No differences between ISDN and nicorandil were found for acute and subacute effects on SVRI, venous hemodynamics, diameter of muscular arteries (FA, BA), and the distensibility and compliance of elastic (CCA) and muscular (FA, BA) arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of reproducibility of a vessel wall movement detector system for assessment of large artery properties.

OBJECTIVE: Arterial distensibility (DC) and compliance (CC) are vessel wall properties of large arteries that can be measured non-invasively with a custom made vessel wall movement detector system (VWMDS). This study investigated the reproducibility of this device in 10 volunteers. METHODS: To investigate intraobserver intrasession and intraobserver intersession variability, arterial diameter (D) and relative change in diameter during the heart cycle (delta D/D) were measured in the elastic common carotid artery, and in the muscular femoral and brachial arteries. Interobserver intrasession variability was examined in common carotid artery by two observers, while interobserver variability on the same image was assessed for common carotid and femoral arteries. Variability was expressed as the coefficient of variation. RESULTS: For common carotid artery, intraobserver intrasession variability was 7.9(SEM 1.6)% (delta D/D), 4.5(1.1)% (D), 8.3(1.3)% (DC), and 9.1(2.6)% (CC), respectively. In femoral artery it was 12.4(2.2)% (delta D/D), 2.7(0.6)% (DC), 13.4(2.2)% (DC), and 12.5(2.1)% (CC). For brachial artery it was 13.4(2.8)% (delta D/D), 2.5(0.5)% (D), 16.1(2.5)% (DC), and 15.6(2.6)% (CC). Intraobserver intersession variability was comparable to intraobserver intrasession variability for all vessels. Interobserver intrasession variability for common carotid artery was 11.3(2.6)% (delta D/D) and 8.6(1.9)% (D), but was larger for DC and CC. Interobserver variability on the same image was < 5% for common carotid and femoral arteries. CONCLUSIONS: In conclusion, the vessel wall movement detector system has a good technical reproducibility. Intraobserver intrasession and intersession variability are comparable, and are larger in muscular arteries. This might be due to a larger variation in tone of these arteries, which are under permanent neurohumoral control. Interobserver intrasession variability was larger than intraobserver variability and might be influenced by differences in observers' skill and spontaneous variation in vessel wall properties.

Time-dependent efficacy of antihypertensive agents in spontaneously hypertensive rats.

The efficacy of antihypertensive agents was compared when given at different time points in the circadian rhythm. Spontaneously hypertensive rats (SHRs) were kept on a 12/12-h cycle with lights on/off at 07:00/19:00 h. A computerized system was used to measure intraarterial blood pressure and heart rate continuously. Agents or vehicle were intravenously injected at two time points. One at the beginning of the sleeping period, at which low efficacy was expected (T = 10), and one at T = 16, which is 3 h before the circadian peaks in blood pressure (BP) and heart rate (HR), aimed at reducing the rise in BP and HR at awakening. The hypotensive effect of propranolol, metoprolol, labetalol, prazosin, clonidine, and rilmenidine was greater when injected at T = 16 than at T = 10 (p < 0.05 for propranolol, metoprolol, and rilmenidine). In contrast, the renal vasodilators captopril and tertatolol were more potent after injection at T = 10. Felodipine was equally effective at both time points. Thus, the effects of antihypertensive agents are related to the phase of the circadian rhythm. The data on the sympatholytic agents in general and beta-blockers and centrally acting agents in particular support antihypertensive regimens with timed administrations.

Renal selective N-acetyl-L-gamma-glutamyl prodrugs: studies on the selectivity of some model prodrugs.

1. In this study, a number of structurally different N-acetyl-L-gamma-glutamyl prodrugs were investigated with respect to selective uptake by the kidney in male Wistar rats. 2. All prodrugs were tested in vitro in rat kidney slices and kidney homogenate to study their uptake and conversion. It was found that the prodrugs of para-nitroaniline (agPNA), aminophenyl acetic acid (agAFA), sulphamethoxazole (agSM), sulphadimethoxine (agSDM), propranolol (agPP) and metoprolol (agMP) were accumulated by a probenecid-sensitive carrier. The prodrug of 4'-aminoantipyrine (agAAP) was not accumulated by a probenecid- or buthionine sulphoximine-sensitive carrier. Unlike all other prodrugs, agAAP and agMP were not, or only a very limited extent converted to the parent compound in vitro. 3. agPNA, agAFA and agPP were also investigated in vivo. The tissue distribution of the prodrugs and the parent drugs was established, as was their urinary excretion and pharmacokinetic behaviour. agPNA and agAFA showed selective uptake by the kidney, in contrast to agPP which accumulated in the liver. The distribution of the parent compounds following prodrug administration was as follows: agPNA was found in kidney and plasma: agAFA in kidney only; agPP in liver only. 4. The factors which determine the selectivity of N-acetyl-L-gamma-glutamyl prodrugs are discussed. The main factors are: the transport into the kidney, the conversion rate, the residence time of the prodrug in the kidney and the presence or absence of competition for uptake and conversation by other tissues, e.g. the liver. It is concluded that this prodrug approach offers the possibility of delivering drugs selectively to the kidney, but also that it is not universally applicable.

Compliance and reactivity of the peripheral venous system in chronic intermittent hemodialysis.

A reduced venous compliance and/or inadequate venoconstriction could impair hemodynamics during hemodialysis. Therefore, compliance and reactivity of the peripheral venous system were assessed in hemodialysis patients and controls using strain gauge plethysmography. Reactivity of the venous system towards an efferent sympathetic stimulus was assessed using a cold pressor test. Results showed that venous compliance was reduced in hypertensive hemodialysis patients compared to normotensive dialysis patients (P = 0.013) and normotensive controls (P = 0.004). After one dosage with a directly acting venodilator (nitroglycerin 5 mg s.l.) and 3 days of treatment with an alpha 1-sympathicolytic agent (Doxazosin 2 mg), venous compliance remained unaltered in hypertensive dialysis patients. During the cold pressor test, the blood pressure response, rise in noradrenaline levels and decline in venous compliance were normal in hemodialysis patients. However, their response to the Valsalva manoeuver was significantly impaired (P = 0.011) compared to healthy controls. We conclude that hypertension, not renal failure, causes the reduction of peripheral venous compliance in hemodialysis patients, for which structural factors might be responsible. Despite the existence of autonomous neuropathy, the reaction of the peripheral venous system towards an efferent sympathetic stimulus is intact in hemodialysis patients.

Acute renal vascular response to ACE inhibition in two rat strains with different susceptibility to the development of glomerulosclerosis.

Normotensive male Wistar rats are susceptible to the development of glomerulosclerosis, a process which can be accelerated by partial renal ablation, while normotensive male Wistar Kyoto (WKy) rats are resistant to glomerulosclerosis. Long-term treatment with angiotensin-I-converting enzyme inhibitors prevents the development of glomerulosclerosis. We studied the acute renal vascular response to bolus injections of captopril (1, 3, and 10 mg/kg). Mean arterial blood pressure (MAP) was significantly less in Wistar rats compared to WKy and not influenced by unilateral nephrectomy. Renal vascular resistance (RVR) and the filtration fraction (FF) were significantly greater in sham and unilateral nephrectomy Wistar rats as compared to WKy. Captopril significantly reduced the RVR in all four groups, but at comparable doses, RVR remained greater in the Wistar sham and Wistar rats following unilateral nephrectomy compared to sham and uninephrectomized WKy respectively. Captopril reduced FF in Wistar rats only. These data indicate an enhanced level of activity of the renal renin-angiotensin system in glomerulosclerosis-susceptible Wistar rats compared to glomerulosclerosis-resistant WKy rats, suggesting that vascular reactivity involving the renin-angiotensin system is an important determinant of the genetically determined differences in susceptibility to glomerulosclerosis in these two rats strains. The strain-dependent difference in RVR at the highest dose of captopril indicates that additional, possibly structural features may play a role in the difference in susceptibility to glomerulosclerosis.

DNA synthesis in the non-infarcted cardiac interstitium after left coronary artery ligation in the rat: effects of captopril.

Effects of Captopril. Journal of Molecular and Cellular Cardiology (1991) 23, 1245-1253. This study was undertaken to investigate the alterations in interstitial DNA synthesis and collagen content in the non-infarcted left and right ventricle after induction of a myocardial infarction (MI) in the rat. MI was induced by ligation of the left anterior descending coronary artery. All animals received 5-Bromo-2'-deoxyUridine (BrdU), via a subcutaneous osmotic minipump, one day before sacrifice, to quantitate DNA synthesis. A transient rise in BrdU incorporation was found in both ventricles. Peak levels were found at day 7 and 14 after infarct induction. BrdU incorporation had returned to control levels 3 weeks after infarct induction. By using anti BRDU--anti-laminin immuno- double staining DNA synthesis was localized mainly in the cardiac interstitium. Concomitantly, a sustained increase in collagen content, measured as the Sirius red positive area on cross sections, was found from day 7 after infarct induction. No changes were found in sham animals. In the second part of the study the effects of the angiotensin I converting enzyme inhibitor captopril and the arteriolar vasodilator hydralazine on MI induced interstitial DNA synthesis and collagen content were investigated. Captopril reduced both the increase in DNA synthesis and collagen content. Hydralazine did not affect interstitial DNA synthesis, but reduced the MI induced collagen content. Both drugs had no effects in sham animals. We conclude that induction of a myocardial infarction stimulates interstitial DNA synthesis and increases the collagen content in the non-infarcted areas of the heart. Interstitial DNA synthesis is dependent on the angiotensin I converting enzyme in a direct manner independent from afterload changes.

The microcirculation and essential hypertension.

Essential hypertension is characterized by a progressive increase of the mean arterial pressure paralleled by a concomitant increase in the total peripheral resistance. This elevated resistance is the consequence of (a) a decreased internal diameter, (b) an increased wall-to-lumen ratio or (c) a decreased number of small arteries or arterioles. A considerable part of the elevated vascular resistance is determined at the microcirculatory level. This paper reviews the studies performed to unravel the resistance-elevating mechanisms in the microvasculature of different tissues. Furthermore the possible role of the microcirculation in the pathogenesis of essential hypertension is discussed.

In vivo DNA synthesis is not uniformly increased in arterial smooth muscle of young spontaneously hypertensive rats.

We compared the distribution of DNA synthesis over the arterial tree of young normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) with marginally elevated blood pressure. Six-week-old male SHR and WKY rats were therefore infused with 5-bromo-2'-deoxyuridine (BrdUrd) for 2 days and the nuclear incorporation of the thymidine analogue in the media of various arteries was determined by immunohistochemistry. In WKY rats, 2.5% of the arterial smooth muscle nuclei in elastic, muscular and resistance arteries incorporated BrdUrd. In SHR, DNA synthesis was more marked in large arteries than in resistance arteries. It was in addition significantly larger in the aorta, superior mesenteric, renal and femoral arteries of the SHR than in those of the WKY rats. However, nuclear incorporation of BrdUrd in vivo did not differ between SHR and WKY rats in aortic endothelium, carotid arterial smooth muscle, nor in mesenteric or renal resistance arteries. Between 6 and 20 weeks of age, the number of nuclear profiles per media cross-section did not increase in large arteries of WKY rats and SHR. During this period of time, however, carotid artery and thoracic aorta weight and DNA content increased. SHR large arteries gained more DNA than those of WKY rats. These data indicate that DNA synthesis is uniformly distributed over the arterial system in young WKY rats and that DNA synthesis is elevated in the smooth muscle of large arteries of 6-week-old SHR but not in their resistance arteries.

Microvascular actions of calcium channel antagonists.

The microvascular actions of three calcium channel antagonists were studied in intact spontaneously hypertensive rats (SHR) provided with a dorsal striated muscle microcirculatory chamber. Verapamil and the dihydropyridine derivatives nifedipine and felodipine reduced mean arterial blood pressure (MAP) in a dose-dependent manner. They dilated arterioles of different sizes, with the most pronounced effect being on the smallest precapillary arterioles. Venular diameters were not affected by the calcium antagonists. Approximately 60% of the small arterioles showed a rhythmic pattern of vasodilatation and constriction. This pattern of spontaneous vasomotion was completely blocked by the calcium channel antagonists, especially those of the dihydropyridine type. It is concluded that (a) small precapillary arterioles play an important role in the vasodilator action of calcium channel antagonists, and (b) arteriolar vasomotion depends on vascular smooth muscle cell calcium influx.