Capillary rarefaction in treated and untreated hypertensive subjects

Abstract

This study aimed to determine if capillary rarefaction is detectable and associated with endothelial dysfunction in persons with mild systolic blood pressure (SBP) elevation. Capillary density and endothelial function were quantified for 150 nondiabetic participants, grouped by blood pressure (BP) as normotensive, untreated high BP, and treated high BP. Structural capillary rarefaction measures were not different between the three groups. Functional capillary rarefaction measures were significantly lower in both high BP groups compared to normotensives, and correlated inversely with endothelial function. The study findings indicate that the hypertensive vascular pathologic process is already underway at modest levels of blood pressure elevation.

Introduction

Hypertension is a major risk factor for cardiovascular morbidity and mortality [Kannel and Higgins, 1990]. Research to date provides evidence that microvascular rarefaction (reduced number of arterioles and capillaries) is involved in the pathogenesis of hypertension [Feihl et al. 2006; Antonios, 2006; Levy et al. 2001], and it has been proposed that blood pressure elevation may be a consequence of functional and structural changes in the microcirculation [Cohn, 2007]. A reduction in the number of capillaries indirectly reflects structural change in microvessels, which contribute to increased peripheral vascular resistance and an increase in blood pressure [Levy et al. 2001; Vicaut, 1999]. However, whether capillary rarefaction precedes the development of established hypertension or is secondary to the deleterious effects of elevated blood pressure on the microvasculature remains unclear. Additionally, the effect of antihypertensive treatment on capillary rarefaction has not been well established.

Studies in non-black individuals have previously demonstrated the presence of both structural (anatomic absence) and functional (due to nonperfusion) rarefaction, mainly in untreated subjects with moderate-severe hypertension [Debbabi et al. 2006; Serne et al. 2001; Gasser and Buhler, 1992; Antonios et al. 1999a, 1999b]. The primary objective of this study was to determine whether structural and/or functional capillary rarefaction are detectable in untreated patients with high BP (HBP: SBP=130−160 mm Hg) and treated patients with high BP (HBPrx: SBP<160 mm Hg). Since endothelial dysfunction has been implicated in the pathogenesis of hypertension and atherogenesis [Schram et al. 2005; Davignon and Ganz, 2004; Bonetti et al. 2003; Widlansky et al. 2003; Poredos, 2002], another objective was to determine whether there was a relationship of functional or structural capillary rarefaction with endothelial dysfunction.

Research methods and procedures

Study participants

Male and female volunteers, between 18 and 55 years of age, with systolic blood pressure (SBP) less than 160 mm Hg were eligible for enrollment. Exclusion criteria were diabetes, pregnancy, secondary hypertension, coronary or cerebrovascular disease, collagen vascular disease, and organ failure (heart, kidney, liver). The study protocol was approved by the Thomas Jefferson University Institutional Review Board and all participants underwent an informed consent process.

Study design

Participants were interviewed to obtain information on health status and current health behaviors. Height and weight were measured and body mass index (BMI) was computed as weight (in kg) divided by height (in m) squared. Heart rate and two successive blood pressure readings (with a one-minute interval between measurements) were taken from participants in the seated position, following 10 minutes of rest. A Dinamap ProCare 100 automatic blood pressure monitor (GE Healthcare, Piscataway, NJ) with the appropriate size cuff was used on the left arm for all subjects.

Prehypertension is defined in JNC VII as systolic blood pressure (SBP) 120−139 mm Hg or diastolic blood pressure (DBP) 80−89 mm Hg [JNC 7 Express, 2003]. Since prehypertensive subjects with SBP 130−139 mm Hg (high normal) are at increased risk for cardiovascular morbidity and mortality, while prehypertensive subjects with SBP < 130 mm Hg are not [Liszka et al. 2005; Vasan et al. 2001; O'Donnell et al. 1997], we selected 130 mm Hg as the SBP cut-point differentiating subjects with and without mild blood pressure elevation in this study. Therefore, subjects with untreated high normal blood pressure (SBP = 130−139 mm Hg) or Stage 1 hypertension (SBP 140−159 mm Hg) were designated high BP =(HBP). Subjects who were taking antihypertensive medication and had SBP < 160 mm Hg were classified as treated HBP (HBPrx), while subjects with SBP < 130 mm Hg without a medical history of hypertension were classified with normal blood pressure (NBP).

To exclude undiagnosed diabetes, a fasting blood sample for plasma glucose was obtained and assayed for glucose concentration. Individuals with blood glucose of 7.0 mmol/L (126 mg/dL) or higher were excluded from participation. Subjects with a blood glucose value between 6.1−6.9 mmol/L (110−125 mg/dL) were asked to return for a two-hour glucose challenge with a standardized 75 g oral glucose tolerance beverage (Fisher Scientific; Hanover Park, IL). Subjects with two-hour glucose of 11.1 mmol/L (200 mg/dL) or higher were excluded from participation.

Capillary microscopy

The capillaroscopy technique was adapted from Serne and his colleagues [Serne et al. 2001]. Following a minimum 10 hour overnight fast and 20 minutes of seated rest, microvascular measurements were conducted for one hour between 7 and 11 am, in a quiet, temperature-controlled room (maintained between 21.5−22.5°C), with the subject in the seated position and the left hand at heart level. Nailfold capillaries in the dorsal skin of the third finger were visualized using a stereo microscope (Olympus; Center Valley, PA), linked to a 4 megapixel SPOT Insight monochrome digital camera (Model number IN-1400: Diagnostic Instruments; Sterling Heights, MI), and a laptop computer (Dell Latitude D600: Dell; Austin, TX). To limit movement, the left hand and forearm were loosely covered with a folded blanket, and rested on another folded blanket positioned at the base of the microscope. Nailbed illumination was achieved with a 250-W halogen fiber optic lamp (KL 2500 LCD:Schott-Fostec; Elmsford, NY); additional illumination from a supplemental 150W fiber optic halogen light source (B&B Microscopes, Ltd., Warrendale, PA) was used in darkly pigmented individuals. To visualize the capillaries, the 3.2x objective (Olympus 3.2/0.07) was used with a total system magnification of 38.4x. Using SPOT imaging software provided with the camera, light/dark contrast in the capillary photographs was enhanced using the same standard SPOT software function (stretching of bright and dark levels) to maximize visibility of the capillaries in all subjects.

Capillary density was defined as the number of capillaries per square millimeter of nailfold skin, and was computed as the mean of four measurements obtained from the four most clearly focused images, least distorted by movement. Images were counted by individuals blinded to the identity and blood pressure of the study subjects. The reproducibility of the counting procedure was verified with three observers performing capillary counts independently on photographs of different subjects. Following training, subsequent counts performed independently showed a high level of agreement, with group standard deviations ranging from 1.5−4.3 capillaries per square millimeter, for mean capillary counts ranging from 64.0−103.8 capillaries per square millimeter.

To quantify capillary density, digital photomicrographs were taken every 3−5 seconds during each of three stages, at resting baseline, during postocclusive reactive hyperemia, and during venous occlusion. (1) At resting baseline, photomicrographs were taken over a three-minute period to detect capillaries perfused at rest. (2) During postocclusive reactive hyperemia, photomicrographs were taken to quantify functionally perfused capillaries (baseline plus reserve capillaries), as follows. First, an occlusion cuff on the left upper arm was inflated to 40 mm Hg above systolic pressure for 10 minutes. Photomicrographs were then taken during the first minute immediately following release of arterial occlusion, visualizing all functionally perfused capillaries. Lower capillary density following reactive hyperemia indicates impaired functional capillary recruitment, and therefore functional rarefaction. (3) During venous occlusion photomicrographs were taken to quantify maximal capillary density, which includes both perfused (with active red blood cell (RBC) motion) and nonperfused (filled with stagnant, non-moving RBCs) capillaries [Antonios et al. 1999a, 1999b] as follows. Following ten minutes of rest after the postocclusive reactive hyperemia procedure, the arm cuff was inflated to 50 mm Hg for 60 seconds, passively forcing blood into all patent capillaries present and photomicrographs were taken during this time. Since maximal capillary density includes all capillaries structurally present, a reduction in maximal capillary density indicates structural rarefaction.

Table 1 summarizes the capillary density measurements and calculations. Percent capillary recruitment was assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyper-emia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. Lower values for these measures indicate functional capillary rarefaction.

Table 1.

Measures of capillary structure and function.

Capillary density = number of capillaries per square millimeter (mm2) of finger nailfold skin

(A) Resting baseline: continuously perfused capillaries [Serne et al. 2001]

(B) Postocclusive reactive hyperemia: continuously perfused + intermittently perfused (functional reserve) capillaries; measure of capillary function [Serne et al. 2001]

(C) Venous occlusion (maximal capillary density): maximal visualization of all capillaries present, including both perfused (with active red blood cell (RBC) motion) and nonperfused (filled with stagnant, non-moving RBCs) capillaries; measure of capillary structure [Antonios et al. 1999c]

Percent capillary recruitment = (B-A) ÷ C × 100

[Postocclusive reactive hyperemia capillary density – resting baseline capillary density]

÷

Maximal capillary density (during passive venous congestion)

Measure of capillary function

Percent perfused capillaries = (B÷C) × 100

Postocclusive reactive hyperemia capillary density

÷

Maximal capillary density (during passive venous congestion)

Measure of capillary function

Endothelial Function

Endothelial function was assessed before and after postocclusive reactive hyperemia, using non-invasive plethysmography measurements of forearm blood flow, according to the method of Sivertsson [1970], which utilizes the endothelium-dependent stimulus of reactive hyperemia to induce vasodilation. With the subject in the seated position following 10 minutes of supine rest, a mercury-in-rubber strain gauge stretched to 10% beyond its resting length was looped around the subject's forearm 5 cm below the antecubital fossa. The strain-gauge was connected to a plethysmograph (EC-4: DE Hokanson, Inc; Bellevue, WA), which in turn was connected to a Doppler recorder (CW-1; DE Hokanson, Inc; Belleveue, WA). An upper arm occlusion cuff was applied, and the arm was suspended comfortably at heart level using a sling bandage connected to an adjustable intravenous pole. Systolic and diastolic blood pressures and heart rate were obtained with a Dinamap cuff placed on the opposite arm. A pediatric cuff around the wrist was inflated to 200 mm Hg to occlude flow to the hand. The upper arm cuff was inflated to 50 mm Hg, deflated for 1.5 seconds, and then re-inflated rapidly prior to each forearm blood flow measurement, obtained through expansion of the strain gauge placed around the forearm. Forearm blood flow (FBF) was measured at rested baseline (FBF_base) and again at postocclusive hyperemia-induced maximal vasodilation (FBF_max). For baseline blood flow measurements, four consecutive FBF curves were obtained within 30 seconds (FBF_base). The occlusion cuff was then inflated to 40 mm Hg above systolic pressure for 10 minutes. Following release of arterial occlusion (postocclusive reactive hyperemia), four consecutive FBF curves were obtained within the first 30 seconds of flow (FBF_max).

The ratio FBF_max/FBF_base was computed as an estimate of vasodilation, by dividing the mean of the four FBF_max values by the mean of the four FBF_base values [Raitakari and Celermajer, 2000]. Forearm vascular resistance at maximal vasodilation (FVR_max) was calculated as the mean arterial pressure (MAP) divided by FBF_max. FBF_max during reactive hyperemia is directly related to FBF after maximum infusion of intra-arterial acetylcholine, an endothelial-dependent vasodilator [Pasimeni et al. 2006]. Accordingly, FBF_max and the ratio FBF_max/FBF_base are accepted non-invasive measures of endothelial function [Tousoulis et al. 2005; Higashi and Yoshizumi, 2003; Higashi et al. 2001]. In addition, both FBF_max and FVR_max reflect resistance artery structural changes (increased wall/lumen ratio) [Lind et al. 1998].

Statistical analyses

Correlations between SBP, DBP, and vascular measures (capillary densities, percent capillary recruitment, percent perfused capillaries, ratio FBF_max/FBF_base and FVR_max) were computed with Spearman's rank-order correlation coefficient. Linear regression analysis was then performed to compare the three separate groups (normotensives, NBP; treated hypertensives, HBPrx; and untreated hypertensives, HBP) with respect to the vascular measures noted above, controlling for age, gender, race, and BMI. P-values < 0.05 were considered significant. Analyses were performed using SAS 9.1 (SAS Institute; Cary, NC).

Results

Data were obtained on a total sample of 150 subjects (93 females and 57 males), ages 19−55 years. The racial/ethnic composition of the study population was 52% non-black (42% Caucasian, 7% Hispanic, and 3% Asian) and 48% black. Descriptions of the enrolled study subjects, together with mean values for the vascular measurements, are shown in Table 2. The BP groups consisted of 91 NBP, 24 HBP, and 35 HBPrx. Among the 35 patients in the HBPrx group, diuretics were the most commonly prescribed antihypertensives (N = 21; 12 as monotherapy), followed by ACE inhibitors (N = 10; 7 as monotherapy), beta blockers (N = 7; 3 as monotherapy), calcium channel blockers (N = 6; 2 as monotherapy), and angiotensin receptor blockers (N = 3; 2 as monotherapy).

Table 2.

Study participant characteristics (N = 150).

	NBP* (N = 91)	HBPrx* (N = 35)	HBP* (N = 24)
Age (years), mean ± sd	37 ± 9	41 ± 9	38 ± 10
Gender, n (%)
Female	55 (60)	27 (77)	11 (46%)
Male	36 (40)	8 (23)	13 (54%)
Race, n (%)
Black	39 (43)	19 (54)	14 (58)
Non-black**	52 (57)	16 (46)	10 (42)
BMI (kg/m2), mean ± sd	27.9 ± 6.7	33.5 ± 8.0	32.1 ± 7.1
BMI (kg/m2), n (%)
Normal weight (18.5 ≤ BMI ≤ 24.9)	36 (39)	3 (9)	3 (13)
Overweight (25.0 ≤ BMI ≤ 29.9)	29 (32)	14 (40)	7 (29)
Obese (30.0 ≤ BMI)	26 (29)	18 (51)	14 (58)
SBP (mm Hg), mean ± sd	114 ± 9	129 ± 10	138 ± 8
DBP (mm Hg), mean ± sd	70 ± 7	76 ± 8	79 ± 10
Capillary densities (capillaries/mm2), mean ± sd
Baseline	55 ± 15	52 ± 15	58 ± 19
Postocclusive reactive hyperemia	67 ± 17	59 ± 16	66 ± 20
Venous occlusion (maximal)	72 ± 18	71 ± 19	78 ± 20
% Capillary recruitment, mean ± sd	16.5 ± 7.1	9.5 ± 5.2	9.9 ± 4.8
% Perfused capillaries, mean ± sd	92.5 ± 5.3	82.6 ± 7.2	83.9 ± 9.0
FBFmax/FBFbase, mean ± sd	9.2 ± 3.1	5.9 ± 2.4	6.9 ± 2.2
FVRmax, mean ± sd	3.3 ± 1.6	5.9 ± 4.3	4.8 ± 2.0

^*NBP: normal blood pressure; HBPrx: high blood pressure, treated; HBP: high blood pressure, untreated.

^**Mostly Caucasians (N = 62), but also including Hispanics (N = 11) and Asians (N = 5).

Table 3 summarizes the Spearman correlations between capillary density measures and the other vascular measurements as continuous variables, within the entire study sample. Capillary density measurements (baseline, postocclusive, and maximal) did not correlate significantly with either SBP or DBP. There was a significant inverse relationship between the percent perfused capillaries with both SBP and DBP (p<0.001 for both), and between percent capillary recruitment and SBP (p = 0.003). Both measures of capillary function (percent capillary recruitment and percent perfused capillaries) correlated with the plethysmographic measures (ratio of FBFmax/FBFbaseline and FVRmax: all p < 0.001).

Table 3.

Spearman correlation coefficients between blood pressure, capillary measures, and vascular measures.

		Blood pressure		Endothelial function	Microvascular structure
		SBP	DBP	FBFmax/FBFbase	FVRmax
Blood pressure	SBP		0.643 (p < 0.001)	−0.301 (p < 0.001)	0.344 (p < 0.001)
	DBP	0.643 (p < 0.001)		−0.221 (p = 0.007)	0.270 (p < 0.001)
Capillary densities	Baseline	−0.066 (p = 0.420)	−0.135 (p = 0.100)	0.034 (p = 0.680)	0.0026 (p = 0.975)
	Postocclusive reactive hyperemia	−0.146 (p = 0.075)	−0.156 (p = 0.057)	0.101 (p = 0.217)	−0.152 (p = 0.063)
	Venous occlusion (maximal)	−0.022 (p = 0.786)	−0.025 (p = 0.765)	0.034 (p = 0.677)	−0.029 (p = 0.721)
%Capillary recruitment		−0.245 (p = 0.003)	−0.080 (p = 0.329)	0.449 (p < 0.001)	−0.440 (p < 0.001)
% Perfused capillaries		−0.459 (p < 0.001)	−0.300 (p < 0.001)	0.395 (p < 0.001)	−0.398 (p < 0.001)

In further linear regression analyses, we compared the capillary and plethysmographic measures across the three blood pressure groups (NBP, HBPrx, and HBP). Table 4 summarizes the results of both the unadjusted and adjusted analyses (adjusted for age, gender, race, and BMI) among the three blood pressure groups. The adjusted differences were slightly attenuated compared to the unadjusted results, but retained statistical significance.

Table 4.

Regression results for capillary and vascular measures (unadjusted and adjusted for age, gender, race and BMI).

	Unadjusted			Adjusted
	Overall	HBPrx vs. NBP*	HBP vs.NBP*	Overall	HBPrx vs. NBP*	HBP vs. NBP*
Baseline capillary density
Mean difference (95%CI)		−2.9 (−9.1, 3.3)	3.4 (−3.7, 10.5)		−3.7 (−10.3, 2.9)	4.3 (−3.1, 11.8)
p	0.320	0.353	0.351	0.438	0.268	0.254
Postocclusive reactive hyperemia capillary density
Mean difference (95%CI)		−8.4 (−15.1, −1.6)	−0.91 (−8.7, 6.9)		−8.4 (−15.6, −1.2)	0.57 (−7.6, 8.8)
p	0.051	0.016	0.817	0.160	0.023	0.891
Venous occlusion (maximal) capillary density
Mean difference (95%CI)		−1.3 (−8.6, 6.0)	5.8 (−2.7, 14.2)		−2.3 (−10.1, 5.5)	5.6 (−3.2, 14.5)
p	0.316	0.726	0.178	0.527	0.568	0.211
% Capillary recruitment
Mean difference (95%CI)		−7.0 (−9.5, −4.5)	−6.6 (−9.4, −3.7)		−5.7 (−8.4, −3.1)	−5.7 (−8.7, −2.7)
p	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
% Perfused capillaries
Mean difference (95%CI)		−9.9 (−12.4, −7.4)	−8.6 (−11.5, −5.7)		−8.7 (−11.2, −6.2)	−6.5 (−9.3, −3.6)
p	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Endothelial function: FBFmax/FBFbase
Mean difference (95%CI)		−3.2 (−4.3, −2.1)	−2.3 (−3.6, −1.0)		−2.5 (−3.6, −1.3)	−1.8 (−3.1, −0.6)
p	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Microvascular structure: FVRmax
Mean difference (95%CI)		2.6 (1.6, 3.6)	1.5 (0.3, 2.6)		2.0 (1.0, 3.0)	1.3 (0.2, 2.5)
p	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

^*NBP: normal blood pressure; HBPrx: high blood pressure, treated; HBP: high blood pressure, untreated.

The three capillary density measures (resting baseline, postocclusive reactive hyperemia, and maximal) did not differ significantly across the three groups (adjusted overall p = 0.438, 0.160, and 0.527 for the three measures, respectively, Table 4). However, the two functional capillary measures (percent capillary recruitment and percent perfused capillaries) were significantly different across the three groups (adjusted overall p < 0.001 for both measures). Compared to the NBP participants, both the HBPrx and HBP groups had significantly lower functional capillary measures (all adjusted p-values <0.001), although they did not differ significantly from each other (p = 0.993 for percent capillary recruitment, and 0.164 for percent perfused capillaries). A comparable pattern was seen for the two plethysmography measures (FBF_max/FBF_base and FVR_max). These also differed significantly between the three groups (adjusted overall p = 0.001 for both measures), with both the HBPrx and the HBP groups being significantly different from the NBP group (all adjusted p-values < 0.001), but not significantly different from each other (p = 0.379 and 0.448 for the two measures respectively).

Discussion

Direct measurement of capillary density by capillaroscopy identified functional capillary rarefaction in both treated and untreated subjects with mild blood pressure elevation, compared to normotensives. There was no significant reduction in baseline or maximal capillary density in either the treated or untreated high BP groups compared to normotensives, indicating the absence of structural capillary rarefaction. These results persisted even after controlling for participant characteristics (including BMI).

Both structural and functional capillary rarefaction have been reported in older individuals with higher blood pressure levels. The current study specifically enrolled younger subjects without severe hypertension to determine if capillary rarefaction, whether structural and/or functional, could be detected in early-stage disease. In this study, the mean age of subjects was approximately 40 years, compared to 45−55 years in previous studies [Debbabi et al. 2006; Antonios et al. 1999a, 1999b], and mean SBP values for hypertensive subjects were 130−140 mm Hg, compared to 155−165 mm Hg in previous studies [Serne et al. 2001; Antonios et al. 1999a, 1999b; Gasser and Buhler, 1992].

In human subjects, some previous reports have described microvessel rarefaction in early stages of hypertension, or even prior to its development among high risk individuals [Antonios et al. 2003; Antonios et al. 1999c; Noon et al. 1997]. However, the progression of capillary rarefaction, and the temporal relationship between capillary rarefaction and hypertension have not been clearly determined. This study's finding of the presence of functional capillary rarefaction in the absence of structural capillary rarefaction is consistent with the theory of Prewitt et al. [1982], that hypertension-induced vasoconstriction leads initially to reversible, functional rarefaction (nonperfusion of capillaries), later followed by irreversible structural rarefaction (anatomic absence of capillaries). According to this theory, younger individuals with mild BP elevation (like those enrolled in this study) would be expected to demonstrate primarily reversible, functional rarefaction, while older individuals with higher blood pressure and established hypertension (like many participants in prior studies), would demonstrate irreversible structural rarefaction.

Our data on individuals with mild BP elevation has clinical relevance in light of the reported increase in cardiovascular risk found in prehypertensive individuals in the Framingham cohort. Vasan et al. [2001] reported that risk for cardiovascular events increases along the continuum of blood pressure, beginning at 130 mm Hg. Measures of capillary function (percent capillary recruitment during postocclusive reactive hyperemia and percent perfused capillaries) were significantly lower in both treated and untreated subjects with mild BP elevation compared to NBP. Our results are also consistent with findings from a study in normotensive subjects that described an inverse relationship between 24-hour SBP and percent capillary recruitment [Serne et al. 1999]. Our data suggest the presence of a graded relationship between functional capillary rarefaction and BP in individuals with systolic BP in the prehypertensive-mildly elevated range.

Both measures of functional capillary rarefaction in this study (percent capillary recruitment and percent perfused capillaries) correlated with both a plethysmographic measure of endothelial dysfunction (ratio FBF_max/FBF_base) and forearm vascular resistance at maximal vasodilation (FVR_max), an indirect estimate of anatomic structural vascular change (increase in media/lumen ratio). These findings suggest an association of mild blood pressure elevation with microvascular injury.

Capillary rarefaction and arteriolar narrowing have been detected in the central microcirculation, in retinal vessels. Subsequently, it has been proposed that retinal microvascular changes may be involved in the pathogenesis of hypertension [Ikram et al. 2006; Smith et al. 2004; Norrelund et al. 1994; Wolf et al. 1994], and may be predictive of coronary and cerebrovascular events [Witt et al. 2006; Wang et al. 2003; Wong et al. 2002]. Peripheral microvascular changes may have prognostic significance as well. Rizzoni et al. [2003] reported that an increased media/lumen ratio found in subcutaneous small artery biopsies was a strong prospective predictor of cardiovascular events over an average 5.4 years of follow up in 128 subjects. Further study is needed to determine whether functional capillary rarefaction has clinically significant predictive value.

Functional or structural vascular abnormalities could ultimately serve as targets for antihypertensive therapy. Recently, Cohn [2007] proposed that BP reduction may actually serve as a marker for a favorable effect on the vasculature, with preservation of vascular structure as the primary mechanism for a favorable effect on clinical outcome. However, the longitudinal effect and clinical significance of antihypertensive medication on capillary rarefaction is largely unknown. In the Trial of Preventing Hypertension study (TROPHY), treatment of prehypertension with an angiotensin receptor blocker (ARB) delayed the development of hypertension [Julius et al. 2006]. While the ARB therapy restricted a further rise in blood pressure, there could also have been a direct effect on the microcirculation.

Consistent with a possible protective effect of antihypertensive treatment on rarefaction, Debbabi et al. [2006] found that effectively treated hypertensive subjects had higher maximal skin capillary density compared to untreated hypertensive subjects. The authors speculated that vasodilation and/or reversal of altered angio-genesis resulting from chronic antihypertensive treatment were possible mechanisms underlying the increased number of capillaries. It is possible that the absence of structural capillary rarefaction in our study was due to varying effects of different antihypertensive medications on capillary density. Most hypertensive patients treated with monotherapy in the study conducted by Debbabi et al. [2006] were taking non-diuretics, while most subjects receiving monotherapy in our study were prescribed diuretics, and nondiuretic medication may be more effective than diuretics for reversing capillary rarefaction. Limited studies of ACE inhibitors in animal studies to date indeed suggest a pro-angiogenic effect of these medications, possibly mediated through bradykinins,VEGF, or nitric oxide [Battegay et al. 2007].

If diuretics have limited or no effect on capillary rarefaction, this could explain the failure to find significant differences in structural and functional capillary density measures between treated and untreated hypertensive subjects in this study. The lack of antihypertensive treatment effect on both the functional capillary measures and the plethysmography measures could also have resulted from insufficient treatment time, or longer duration of blood pressure elevation in treated versus untreated hypertensives.

Alternatively, we may have failed to detect evidence of structural rarefaction due to the relatively small sample size. This may be particularly relevant if differences in maximal capillary density are less pronounced between younger normotensive and hypertensive subjects, like those enrolled in this study, compared to capillary density differences between older normotensives and hypertensives, like those enrolled in previous studies. Larger longitudinal investigations are needed to determine the effects of different antihypertensive medication classes on microvessel structure and function. The finding that adjusting for BMI reduces but does not eliminate the correlation between blood pressure and capillary function indicates a contribution of both BMI and blood pressure to capillary function. Further investigations regarding these relationships are also needed.

In this investigation, we elected to study capillaries in the peripheral circulation, which are readily accessible in the fingertips using a simple stereomicroscope, and can easily be studied without the use of intravenous dye injection [Carpentier, 1999]. It has been stated that the capillaroscopy technique is difficult to perform in highly pigmented subjects, with no data on capillary rarefaction in black subjects reported to date [Debbabi et al. 2006; Shore, 2000]. Using the dual light sources and photo-enhancing software described in the methods, we were able to visualize and quantify capillaries in all enrolled subjects, including darkly pigmented blacks.

In conclusion, functional but not structural capillary rarefaction was detected in both treated and untreated patients with mild blood pressure elevation. These results are consistent with the concept that functional capillary rarefaction precedes irreversible structural rarefaction [Prewitt et al. 1982]. Functional changes in capillary density correlate with endothelial dysfunction, and are detectable in blacks as well as non-blacks.