Subcutaneous Small Resistance Arteries Research Articles

PROGNOSTIC SIGNIFICANCE OF THE WALL TO LUMEN RATIO OF RETINAL ARTERIOLES EVALUATED BY ADAPTIVE OPTICS IN HUMAN HYPERTENSION

Objective: Structural alterations in the microcirculation may be considered an important mechanism of hypertension-mediated organ damage. An increased media to lumen ratio of subcutaneous small resistance arteries evaluated with locally invasive techniques (biopsies of subcutaneous fat, micromyography) may predict the subsequent development of cardiovascular events. However, it is not presently known whether structural alterations of retinal arterioles evaluated with a non-invasive approach (adaptive optics) may have a prognostic meaning. Design and method: Two-hundred and thirty-seven subjects were included in the present study. They were normotensive subjects (n = 65, 27,4%) and patients with essential hypertension (n = 163, 68,8%) or primary aldosteronism (n = 9, 3,8%). All subjects were submitted to a non-invasive evaluation or retinal arteriolar morphology (evaluation of the wall/lumen ratio: WLR) by adaptive optics. The subjects were re-evaluated after an average follow-up time of 5.5 years on order to assess the occurrence of death or cardio-cerebrovascular events. Results: Fifty-four total events occurred in the population enrolled, of which 29 were cardio-cerebrovascular event (ischemic or hemorragic stroke, atrial fibrillation, heart failure, coronary artery disease, peripheral artery disease, including aneurism requiring intervention, cardiac valvular disease) while the remaining were events or deaths for any cause, including neoplastic disease. The event-free survival was significantly less in those with a WLR above the median value of the population (0.28) according to Kaplan-Mayer survival curves and multivariate analysis (p < 0.001 by both the Mantel-Cox test and the Breslow test). Subjects with events had a WLR of retinal arterioles significantly greater than those without events. The evidence was confirmed after restricting the analysis to cardiovascular events, excluding events or deaths of neoplastic origin. The average follow-up time of our subjects was 4.6 ± 1.6 years for a total of 1090 patients-years. Patients with cardiovascular events were older and with more elevated levels of circulating glucose. Conclusions: Structural alterations of retinal arterioles evaluated by adaptive optics may predict cardio-cerebral events and total mortality.

ROLE OF INHIBITION OF CGMP-DEPENDENT PROTEIN KINASE (PKG) SIGNALLING ON ANTICONTRACTILE FUNCTION OF PERIVASCULAR ADIPOSE TISSUE

Objective: Perivascular adipose tissue (PVAT) has an anticontractile effect.This capacity is lost in obesity (Circulation 2009) through adipocyte hypertrophy, leading to hypoxia, inflammation and oxidative stress. It has been previously demonstrated that cGMP-dependent protein kinase (PKG) plays a key role in regulating normal PVAT function (Cardiovasc Res. 2014). Aim of the study was to investigate functional responses of small resistance arteries with and without PVAT in normotensive subjects and obese patients, testing the effects of DT-2,(a peptidic highly selective inhibitor of PKG) on contractile response Design and method: We have investigated 9 normotensive severely obese patients (Obese) and 6 hypertensive severely obese patients (Obese-Hyper) undergoing bariatric surgery, as well as 7 normotensive lean subjects (CTRL) undergoing an election surgical intervention.No sign of local or systemic inflammation was present in any subject or patient. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph. A concentration-response curve to noradrenaline (NA, from 10–9 to 10–5Mol/l) was evaluated in vessels with intact prerivascular fat tissue (WF)and in vessels in which perivascular fat tissue was removed (NoF). Concentration response to NA was repeated in small arteries WF and NoF in the presence of DT-2 (125 nM) in both normoxic and hypoxic (95% N2/5%CO2 for 30’)conditions Results: In normoxic conditions the presence of PVAT reduced contractile response to NA in small arteries from CTRL (ANOVA p = 0.04 vs. NoF). The anticontractile effect of PVAT completely disappeared in Obese and Obese-Hyper patients. In CTRL preincubation with DT-2 lead to increase contractile responses to NA in arteries WF (ANOVA p = 0,04 WF+DT-2 vs WF). This effect was more evident during hypoxic conditions after incubation with DT-2 in all groups. No significant effect was observed on contractile response to NA in arteries WF and NoF from Obese and Obese-Hyper patients after incubation of DT-2, in normoxic conditions Conclusions: The anticontractile effect of PVAT is present in normotensive subjects and it is lost after inhibition of PKG signalling. The absence of effect after preincubation of DT-2 on arteries WF from Obese and Obese-Hyper patients suggests a dysregulation of PKG signalling pathways in these groups, which seems essential for its downstream dilator function in small resistance arteries

Changes in extracellular matrix in subcutaneous small resistance arteries of patients with essential hypertension

Background: In the development of hypertensive microvascular remodeling, a relevant role may be played by changes in extracellular matrix proteins. Aim of this study was the to evaluate some extracellular matrix components within the tunica media of subcutaneous small arteries in 9 normotensive subjects and 12 essential hypertensive patients, submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region.Patients and Methods: Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, fibronectin, laminin, transforming growth factor-beta-1 (TGF-β1) and emilin-1 contents within the tunica media were evaluated by immunofluorescence and relative immunomorphometrical analysis (immunopositivity % of area). The total collagen content and collagen subtypes within the tunica media were evaluated using both Sirius red staining (under polarized light) and immunofluorescence assay.Results: Normotensive controls had less total and type III collagen in respect with hypertensive patients. Fibronectin and TGF-β1 tunica media content was significantly greater in essential hypertensive patients, compared with normotensive controls, while laminin and emilin-1 tunica media content was lesser in essential hypertensive patients, compared with normotensive controls. A significant correlation was observed between fibronectin tunica media content and media to lumen ratio.Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, a relevant fibrosis may be detected; fibronectin and TGF-β1 tunica media content is increased, while laminin and emilin-1 content is decreased; these changes might be involved in the development of small resistance artery remodeling in humans.

Microvascular structure as a prognostically relevant endpoint.

Remodelling of subcutaneous small resistance arteries, as indicated by an increased media-to-lumen ratio, is frequently present in hypertensive, obese, or diabetic patients. The increased media-to-lumen ratio may impair organ flow reserve. This may be important in the maintenance and, probably, also in the progressive worsening of hypertensive disease. The presence of structural alterations represents a prognostically relevant factor, in terms of development of target organ damage or cardiovascular events, thus allowing us a prediction of complications in hypertension. In fact, media-to-lumen ratio of small arteries at baseline, and possibly their changes during treatment may have a strong prognostic significance. However, new, non-invasive techniques are needed before suggesting extensive application of the evaluation of remodelling of small arteries for the cardiovascular risk stratification in hypertensive patients. Some new techniques for the evaluation of microvascular morphology in the retina, currently under clinical investigation, seem to represent a promising and interesting future perspective. The evaluation of microvascular structure is progressively moving from bench to bedside, and it could represent, in the near future, an evaluation to be performed in all hypertensive patients, to obtain a better stratification of cardiovascular risk, and, possibly, it might be considered as an intermediate endpoint in the evaluation of the effects of antihypertensive therapy, provided that a demonstration of a prognostic value of non-invasive measures of microvascular structure is made available.

Relationship Between Different Subpopulations of Circulating CD4+ T-lymphocytes and Microvascular Structural Alterations in Humans.

Different components of the immune system, including innate and adaptive immunity (T-effector lymphocytes and T-regulatory lymphocytes-TREGs) may be involved in the development of hypertension. In addition, it was demonstrated in animal models that TREGs may prevent angiotensin II-induced hypertension and vascular injury/inflammation. However, no data are presently available in humans about possible relationships between T-lymphocyte subtypes and microvascular structural alterations. For this purpose, in the present study, we enrolled 24 normotensive subjects and 12 hypertensive patients undergoing an elective surgical intervention. No sign of local or systemic inflammation was present. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph and the media to lumen ratio (M/L) was calculated. In addition, retinal arteriolar structure was evaluated noninvasively by scanning laser Doppler flowmetry. Capillary density in the nailfold, dorsum of the finger, and forearm were evaluated by videomicroscopy. A peripheral blood sample was obtained before surgery for assessment of T-lymphocyte subpopulations by flow cytometry. Significant negative correlations were observed between indices of microvascular structure (M/L of subcutaneous small arteries and wall to lumen ratio of retinal arterioles) and circulating TREG lymphocytes. A direct correlation was observed between M/L of subcutaneous small arteries and circulating Th17 lymphocytes. In addition, total capillary density was correlated with a TREG effector memory subpopulation. Our data suggest that some lymphocyte subpopulations may be related to microvascular remodeling, confirming previous animal data, and opening therapeutic possibilities.

PP.21.05] RELATIONSHIP BETWEEN MICROVASCULAR STRUCTURE AND T REGULATORY LYMPHOCYTES OF SMALL RESISTANCE ARTERIES

Objective: Recently it has been demonstrated a role for adaptive immunity, particularly for T regulatory lymphocytes (Tregs), in the development of hypertension and in preventing of angiotensin II–induced vascular injury and inflammation in animal models (Barhoumi T et al, Hypertension 2011;57:469–476). However, no data are presently available in human beings about possible relationships between Tregs and microvascular structural alterations. Design and method: In the present study we enrolled 11 normotensive subjects and 8 hypertensive patients undergoing an election surgical intervention. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance artery structure was assessed by wire myography and media to lumen ratio (M/L) was calculated. W/L of retinal arterioles was obtained by Scanning Laser Doppler Flowmetry. Functional (basal) and structural (total) microvascular density were studied by capillaroscopy before and after venous congestion. No sign of local or systemic inflammation was present in any subjects or patients. We extracted genomic DNA from small resistance arteries and analyzed methylation status of the FoxP3 gene promoter involved in Treg lymphocytes activation. Unmethylated FoxP3 has been demonstrated to be specific for Treg lymphocytes. A peripheral blood sample was obtained before surgery for routine chemistry Results: Results are summarized in the Table.A significant positive correlation was detected between Tregs in small resistance arteries and basal, total and delta gain capillary density in the forearm, whereas no correlations were observed with small resistance artery M/L and retinal arteriole W/L. In addition, a significant inverse correlation was detected between Treg in subcutaneous small vessels and C reactive protein. Conclusions: Our data suggest that Treg lymphocytes detected in subcutaneous small resistance artery wall are related with capillary density and inversely related with inflammatory markers suggesting a protecting role of Treg also, probably, in terms of angiogenetic properties.

OP.3D.06] RELATIONSHIP BETWEEN DIFFERENT POPULATIONS OF CIRCULATING T LYMPHOCYTES AND MICROVASCULAR OXIDATIVE STRESS

Objective: Both innate and adaptive immune systems may contribute to the pathogenesis of cardiovascular disease and vascular remodeling through inflammation and oxidative stress. Particularly, the balance between Th1 effector lymphocytes (producing interferon-γ) and T regulatory (Treg) lymphocytes, which elicit an anti-inflammatory activity, may be crucial for blood pressure elevation and organ damage development, at least in experimental models. In particular, Tregs have been previously demonstrated to inversely correlate with subcutaneous small resistance artery media to lumen ratio (M/L) and retinal arteriole wall to lumen ratio (W/L) (unpublished data). Therefore, we evaluate the possible relationship between Th1 or Treg lymphocytes and microvascular oxidative stress. Design and method: We enrolled 11 normotensive subjects and 4 hypertensive patients undergoing an election surgical intervention. No sign of local or systemic inflammation was present in any subjects or patients. All patients underwent a biopsy of subcutaneous fat during surgery. A peripheral blood sample was obtained before surgery for assessment of T lymphocyte populations. Lymphocyte phenotype was evaluated by flow cytometry after 5 hour in vitro activation in order to assess Th1 lymphocytes. Superoxide anion production, as index of oxidative stress, was evaluated in the bioptic tissue by florescent dye dihydroethidium and quantified using an image analyzer. Results: Results are summarized in the Table (RTE: recent thymus emigrant; EM: effector memory).A significant positive correlation was observed between Th1 lymphocytes and superoxide anion production in subcutaneous microvessels. Additionally, a significant inverse correlation was observed between microvascular free radical production and Total Treg, RTE and EM Tregs. Conclusions: Our data suggest that Treg lymphocytes may be protective against microvascular damage, probably because of their anti-oxidant properties confirming an involvement of adaptive immune system on microvascular damage.

Resistant hypertension and structural alterations of subcutaneous small resistance arteries

Resistant hypertension and structural alterations of subcutaneous small resistance arteries

Aging and structural alterations of subcutaneous small resistance arteries in hypertensive patients

Aging and structural alterations of subcutaneous small resistance arteries in hypertensive patients

Abstract P256: Structural Alterations of Subcutaneous Small Resistance Arteries in Resistant Hypertension

Background: It is was suggested that, in resistant hypertension, the presence of particularly pronounced microvascular alterations may contribute to explain the relative lack of response to treatment Patients and Methods: We investigated a population of 94 treated essential hypertensive patients. Secondary forms of hypertension were excluded, and in all patients a 24-hour blood pressure monitoring was performed in order to exclude a white coat effect. In all patients, we evaluated small resistance arteries morphology by a wire micromyographic approach. Subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured. We subdived our patents according to the presence or not of resistant hypertension (clinic blood pressure values above 140/90 mm Hg despite administration of three antihypertensive agent including a diuretic and 24-hour blood pressure values >130/80 mm Hg). Sixteen patients had true resistant hypertension, and were compared with the remaining 78 patients with non-resistant hypertension. Results: are summarized in the Table, The two groups were also different in terms of mean age (57±12 vs. 67±7 years, p=0.016 and pulse pressure/stroke volume, a rough index of large artery distensibility: 0.63±0.31 vs. 0.90±0.33, p=0.02). Conclusion: Hypertensive patients with true resistant hypertension have greater microvascular structural alterations compared with non-resistant hypertensive patients. This could partly explain the resistance to treatment and the high cardiovascular risk observed in these patients.

Abstract P255: Age and Structural Alterations in Subcutaneous Small Resistance Arteries of Hypertensive Patients

Background: It was proposed that early vascular ageing may be an important mechanism of vascular damage in large conductance arteries. However it is not known whether aging may also affect small resistance artery morphology. Patients and Methods: For this reason, we investigated 100 patients with essential hypertension. Secondary forms of hypertension were excluded according to standard clinical evaluations and biochemical or instrumental assessments. In all patients, an evaluation of small resistance arteries morphology was performed by a wire micromyographic approach (Mulvany’s technique). A small amount of subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured. Results: The age range of our populaton was 22-81 years, with a mean value of 57±12 years; 14% of them were current smokers, 32% had alterations in lipid patterns, none of them had diabetes mellitus, 58 were males and average blood pressure values were 156/95±19/12 mmHg. We found a significant correlation between M/L and age (r=0.30, p=0.002): the statistical significance of the correlation persisted after correction for counfounding variables (gender, serum cholesterol, smoking status, serum glucose, systolic or diastolic blood pressure values). A statistically significant inverse correlation was also observed between internal diameter and age (r=-0.20, p=0.046), while the correlation beween age and media thickness did not reach statistical significance (r=0.09, p=0.37). Conclusion: Our data suggest that age may affect microvascular structure in hypertensive patients. It is also possible that hypertension may anticipate the effects of physiological aging, and this should be explored in a relatively large population of normotensive subjects.

7D.03

Objective: It was proposed that early vascular ageing may be an important mechanism of vascular damage in large conductance arteries. However it is not known whether aging may also affect small resistance artery morphology. Design and method: For this reason, we investigated 100 patients with essential hypertension. Secondary forms of hypertension were excluded according to standard clinical evaluations and biochemical or instrumental assessments. In all patients, an evaluation of small resistance arteries morphology was performed by a wire micromyographic approach (Mulvany's technique). A small amount of subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured. Results: The age range of our population was 22–81 years, with a mean value of 57 ± 12 years; 14% of them were current smokers, 32% had alterations in lipid patterns, none of them had diabetes mellitus, 58 were males and average blood pressure values were 156/95 ± 19/12 mmHg. We found a significant correlation between M/L and age (r = 0.30, p = 0.002): the statistical significance of the correlation persisted after correction for counfounding variables (gender, serum cholesterol, smoking status, serum glucose, systolic or diastolic blood pressure values). A statistically significant inverse correlation was also observed between internal diameter and age (r = -0.20, p = 0.046), while the correlation between age and media thickness did not reach statistical significance (r = 0.09, p = 0.37). Conclusions: Our data suggest that aging may affect microvascular structure in hypertensive patients. It is also possible that hypertension may anticipate the effects of physiological aging, and this should be explored in a relatively large population of normotensive subjects.

Effects of a long-term treatment with aliskiren or ramipril on structural alterations of subcutaneous small-resistance arteries of diabetic hypertensive patients.

Structural alterations of subcutaneous small-resistance arteries are associated with a worse clinical prognosis in hypertension and non-insulin-dependent diabetes mellitus. The effects of the direct renin inhibitor aliskiren on microvascular structure were never previously evaluated. Therefore, we investigated the effects of aliskiren in comparison with those of an extensively used angiotensin-converting enzyme inhibitor, ramipril, on peripheral subcutaneous small-resistance artery morphology, retinal arteriolar structure, and capillary density in a population of patients with non-insulin-dependent diabetes mellitus. Sixteen patients with mild essential hypertension and with a previous diagnosis of non-insulin-dependent diabetes mellitus were included in the study. Patients were then randomized to 1 of the 2 active treatments (aliskiren 150 mg once daily, n=9; or ramipril 5 mg once daily, n=7). Each patient underwent a biopsy of the subcutaneous fat from the gluteal region, an evaluation of retinal artery morphology (scanning laser Doppler flowmetry), and capillary density (capillaroscopy), at baseline and after 1 year of treatment. Subcutaneous small arteries were dissected and mounted on a pressurized micromyograph, and the media-to-lumen ratio was evaluated. A similar office blood pressure-lowering effect and a similar reduction of the wall-to-lumen ratio of retinal arterioles were observed with the 2 drugs. Aliskiren significantly reduced media-to-lumen ratio of subcutaneous small-resistance arteries, whereas ramipril-induced reduction of media to lumen ratio was not statistically significant. No relevant effect on capillary density was observed. In conclusion, treatment with aliskiren or ramipril was associated with a correction of microvascular structural alterations in patients with non-insulin-dependent diabetes mellitus.

Anatomical remodelling of the anterior abdominal wall arteries in obesity

Obesity is associated with structural alterations in subcutaneous small resistance arteries. The aim of the present work is to study modifications of perforators vessels of abdominal wall and subcutaneous tissue characteristics in obese patients after massive weight loss. An anatomo-radiologic study was carried out on 15 patients (5M, 10F, mean age 54.9 y), who underwent abdominoplasty after massive weight loss. Their pre-operative Computed Tomographic (CT) results of the anterior abdominal wall were compared with CT of 15 normal weighted controls. Anatomo-microscopic and morphometric examinations were conducted on full-thickness specimens of panniculectomy samples. 10 right panniculectomy were sampled from donor cadavers. All the measurements were taken on transverse sections. In patients, at CT the mean luminal diameter (LD) and standard deviation of perforator branches of the deep inferior epigastric artery (DIEA) was 3.7 ± 0.4 mm (control 2.2 ± 0.1 mm; p < 0.05). At microscopic examination, the wall thickness of perforator arteries was 212.7 μ ± 83.9 versus 143.9 ± 32.8 (p < 0.05) deep to the superficial fascia and 120.4 μ ± 74.8 versus 72.3 ± 23.5 (p < 0.05) superficial to it. A thickening of the muscular layer was observable and the tunica media represented 71.4% ± 5.6 of the whole area of the wall (controls 37.1% ± 3.5, p < 0.0001). Our data demonstrate that the major LD of the perforators in patients matches with hypertrophy of the tunica media and we think that the major thickness of perforator walls can facilitate the microsurgical technique in free microsurgical flap reconstruction.

Abstract 379: Circulating T Regulatory Lymphocytes and Microvascular Structural Alterations in Hypertensive Patients and Normotensive Subjects

Background and Methods. Different components of the immune system, including innate immunity and adaptive immunity (T effector lymphocytes and T regulatory lymphocytes: Tregs) may be involved in the development of hypertension. In addition, it was recently demonstrated that Tregs may prevent angiotensin II-induced vascular injury/inflammation, while Th1, Th2 and Th17 may play a role in the progression of vascular remodeling. However, no data are available in human beings about relationships between T-lymphocyte subtypes and microvascular structure. In the present, preliminary study, we enrolled 7 normotensive subjects and 3 hypertensive patients undergoing an election surgical intervention. No sign of local or systemic inflammation was present in any subjects or patients. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph, morphological parameter were measured, in particular media to lumen ratio (M/L) was calculated. A peripheral blood sample was obtained before surgery for assessment of T lymphocytes subpopulations by flow cytometry. Results are summarized in the Table. RTE: recent thymus emigrant; naïve: no contact with antigens, TDEM: highly antigen-experienced cells that assume pro-apoptotic properties. Additional significant positive correlations were observed between M/L and Th17 effector lymphocytes: r=0.67, p&lt;0.05). Conclusion Our data suggest that some lymphocytes subpopulations may be related to microvascular remodeling, and open the possibility to regress small artery remodeling in human by specific drug modulation of lymphocyte subtypes.

Circulating endothelial progenitor cells, microvascular density and fibrosis in obesity before and after bariatric surgery

It is not known whether, in obesity, the capillary density or the number of circulating endothelial progenitor cells (EPCs) are reduced, or whether fibrosis of small vessels is also present. In addition, possible effects of weight reduction on these parameters have never been evaluated. Therefore, we investigated EPCs and capillary density in 25 patients with severe obesity, all submitted to bariatric surgery, and in 18 normotensive lean subjects and 12 hypertensive lean patients as controls. All patients underwent a biopsy of subcutaneous fat during bariatric surgery. In five patients, a second biopsy was obtained after consistent weight loss, about 1 year later, during a surgical intervention for abdominoplasty. EPCs and capillary density were reduced in obesity, and EPCs were significantly increased after weight reduction. Vascular collagen content was clearly increased in obese patients. No significant difference in vascular collagen was observed between normotensive obese patients and hypertensive obese patients. After pronounced weight reduction, collagen content was nearly normalized. No difference in stress–strain relation was observed among groups or before and after weight loss. In conclusion, our data suggest that microvascular rarefaction occurs in obesity. EPCs were significantly reduced in obese patients. Pronounced weight loss induced by bariatric surgery seems to induce a significant improvement of EPC number, but not of capillary rarefaction. A pronounced fibrosis of subcutaneous small resistance arteries is present in obese patients, regardless of the presence of increased blood pressure values. Consistent weight loss induced by bariatric surgery may induce an almost complete regression of microvascular fibrosis.

Relationship between media-to-lumen ratio of subcutaneous small arteries and wall-to-lumen ratio of retinal arterioles evaluated noninvasively by scanning laser Doppler flowmetry

Structural alterations of subcutaneous small resistance arteries, as indicated by an increased media-to-lumen ratio, are frequently present in hypertensive and/or diabetic patients, and may represent the earliest alteration observed. Furthermore, media-to-lumen ratio of small arteries evaluated by micromyography has a strong prognostic significance; however, its extensive evaluation is limited by the invasivity of the assessment, since a biopsy of subcutaneous fat is needed. Noninvasive measurement of wall-to-lumen of retinal arterioles using scanning laser Doppler flowmetry (SLDF) has recently been introduced. However, this new technique has not yet been compared to micromyographic measurement, generally considered the gold standard approach. We investigated 40 individuals and patients, 24 of them were hypertensive patients and 16 normotensive individuals. All patients underwent a biopsy of subcutaneous fat during an elective surgical intervention. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph, and media-to-lumen ratio was measured. In addition, an evaluation of wall-to-lumen ratio of retinal arterioles by SLDF was performed (Heidelberg Retina Flowmeter, Heidelberg Engineering). A close correlation was observed between media-to-lumen ratio of subcutaneous small arteries and wall-to-lumen ratio of retinal arterioles (r = 0.76, P < 0.001; P < 0.001, r(2) = 0.57). A noninvasive and easily repeatable procedure (intraobserver and interobserver variation coefficient <13%) such as an evaluation of the arterioles in the fundus oculi by SLDF may provide similar information regarding microvascular morphology compared with an invasive, accurate and prognostically relevant micromyographic measurement of media-to-lumen ratio of subcutaneous small arteries.

How to Evaluate Microvascular Organ Damage in Hypertension

Patients with essential hypertension are characterized by endothelial dysfunction and impaired nitric oxide availability secondary to oxidative stress production. A dysfunctioning endothelium is an early marker of the development of atherosclerotic changes and can also contribute to cardiovascular events. The autocrine/paracrine activity of endothelial cells makes it very difficult to investigate endothelial function in clinical research. Vascular reactivity tests represent the most widely used methods of clinical assessment of endothelial function. The aim of these tests is to activate or block endothelial cell function while measuring consequent changes in vascular tone in selected vascular districts. Many studies have evaluated the endothelium in hypertensive patients, using different techniques. Several methodologies were developed to study microcirculation (resistance arteries and arterioles), both in coronary and peripheral vascular districts. In particular, peripheral microcirculation can be evaluated in functionally isolated vascular districts such as the peripheral muscle (usually forearm), the subcutaneous tissue, or the skin. This article focuses on the most relevant available techniques in the research on endothelial dysfunction in essential hypertension, in particular their advantages and major limitations. The coronary microcirculation can be evaluated by measuring coronary blood flow with Doppler flow wire and quantitative angiography during the intracoronary infusion of endothelial agonists. The isolated and perfused forearm technique is the most often used approach in the assessment of endothelial function in peripheral muscle microcirculation. The subcutaneous small resistance arteries can be studied using the Mulvany myograph device, an in vitro, ex vivo technique. Finally, the use of the laser digital Doppler technique provides a non-invasive approach to the evaluation of the skin microcirculation. Each technique employed to assess endothelial function has specific limitations that hinder its applicability in large-scale screening. In addition, studies assessing the degree of correlation among measures of endothelial function, as evaluated in different vascular beds, have demonstrated a poor relationship and, while statistically significant, the findings are of no clinical relevance. Therefore, no one technique can be considered a surrogate for another. At the present time, the optimal methodology for investigating the multifaceted aspects of endothelial dysfunction is still under debate. Only the growing concordant results from different reproducible and reliable methods exploring endothelial function with different stimuli will support and strengthen experimental findings, thus providing conclusive answers in this area of research.

How to Assess Microvascular Structure in Humans

Structural alterations of subcutaneous small resistance arteries, as indicated by an increased media to lumen ratio, are frequently present in hypertensive and/or diabetic patients. However, the evaluation of microvascular structure is not an easy task. Among the methods that may be applied to humans, plethysmographic evaluation of small arteries and wire or pressure micromyography were extensively used in the last decades. Media to lumen ratio of small arteries evaluated by micromyography was demonstrated to possess a strong prognostic significance; however, its extensive evaluation is limited by the invasiveness of the assessment, since a biopsy of subcutaneous fat is needed. Non-invasive approaches were then proposed, including capillaroscopy, which provides information about microvascular rarefaction. Recently, the interest of investigators has focused on the retinal microvascular bed. In particular, a non-invasive measurement of wall thickness to internal lumen ratio of retinal arterioles using scanning laser Doppler flowmetry has been recently introduced. Preliminary data suggest a fairly good agreement between this approach and micromyographic measurements, generally considered the gold standard approach. Therefore, the evaluation of microvascular structure is progressively moving from bench to bedside, and it could represent, in the immediate future, an evaluation to be performed in all hypertensive patients, in order to obtain a better stratification of cardiovascular risk.

Investigation of the effects induced on endothelial function by the human T2238C ANP gene molecular variant

s 153 Clinica Medica, Department of Medical and Surgical Sciences, University of Brescia, Brescia, Italy Clinica Chirurgica, Department of Medical and Surgical Sciences, University of Brescia, Brescia, Italy Chair of Ophthalmology, University of Brescia, Brescia, Italy Chair of Ophthalmology, University of Molise, Campobasso, Italy Chair of Ophthalmology, University of Ferrara, Ferrara, Italy Structural alterations of subcutaneous small resistance arteries, as indicated by an increased media to lumen (M/L) ratio, are frequently present in hypertensive and/or diabetic patients, and may represent the earliest alteration observed. In addition, M/L of small arteries evaluated by micromyography has a strong prognostic significance; however its extensive evaluation is limited by the invasivity of the assessment, since a biopsy of subcutaneous fat is needed. Non-invasive measurement of wall to lumen (W/L) of retinal arterioles using scanning laser doppler flowmetry (SLDF) has been recently introduced (Harazny J et al, Hypertension 2007; 50:623-629). However, this new technique was never compared with micromyographic measurement, considered the gold standard approach. Methods and Results: We have investigated 37 subjects and patients. Twenty-three of them were hypertensives and 14 normotensives. Blood pressure values were 139/88 17/10 mmHg and 125/71 17/12 mm Hg, respectively (p<0.05). All patients underwent a biopsy of subcutaneous fat during an elective surgical intervention. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph, and M/L was measured. In addition, an evaluation of W/L of retinal arterioles by SLDF was performed (Heidelberg Retina Flowmeter, Heidelberg Engineering). A close correlation was observed between M/L of subcutaneous small arteries and WL of retinal arterioles: rZ0.77, p<0.001 (see Figure). Conclusion: A non invasive and easily repeatable procedure (intraobserver and interobserver variation coefficient around 10%) such as an evaluation of the arterioles in the fundus oculi by SDLF may provide similar information regarding microvascular morphology compared in respect with an invasive, accurate and prognostically relevant micromyographic measurement of M/L. y = 6,5298x 0,234 R2 = 0,5902 0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,04 0,06 0,08 0,10 0,12 0,14 0,16