C. De Ciuceis

Decreased number of circulating endothelial progenitor cells in patients with Graves’ hyperthyroidism

Objective: A relevant biological role of circulating endothelial progenitor cells (EPC) was recently demonstrated. EPC are generated in the bone marrow, and interact with damaged endothelium, restoring the integrity of the monolayer. Therefore, aim of the present study was to evaluate EPC in the blood of patients with untreated Graves’ hyperthyroidism (GD), in whom an increased oxidative stress was observed. Design and methods: Twenty-three patients with untreated active GD and 18 matched normal controls (NC) were included in the study. Circulating EPC were isolated from peripheral blood. Mononuclear cells were cultured with endothelial basal medium supplemented with EGM SingleQuots, and were identified by positive double staining after 7 days in culture. Circulating levels of C reactive protein, total antioxidant power, interleukin (IL)-6, IL-18, monocyte chemoattractant protein-1, tumor necrosis facotr-α, soluble vascular cell adhesion molecule (VCAM) and intracellular adhesion molecule were evaluated by enzyme-linked immunosorbent assay kit. EPC number was also evaluated in a subgroup of GD patients after restoration of euthyroidism. Results: Systolic blood pressure resulted increased in GD patients compared with control subjects whereas diastolic blood pressure was not significantly different. Patients with GD showed an increase in circulating levels of IL-18 and VCAM-1 and a reduction of total antioxidant power (p<0.05) compared to NC. Moreover, a reduced number of EPC was observed in patients with GD compared to NC (p<0.05) which turned to NC values after restoring euthyroidism. Conclusion: Patients with GD showed a reduction in the physiological protective mechanisms against endothelial damage, probably induced by increased inflammation and oxidative stress.

[PP.12.08] EFFECTS OF MELATONIN ON THE PRODUCTION OF ADIPONECTIN AND THE EXPRESSION OF ADIPONECTIN RECEPTOR IN THE VISCERAL ADIPOSE TISSUE OF AGING MICE.

Objective: It has been previously demonstrated that inflammation in adipose tissue may be implicated in vascular dysfunction (Circulation 2009; 119(12):1661–1670). A senescence-accelerated prone mouse (SAMP8) is a model of age-related cognitive decline and vascular dysfunction. Several studies demonstrated that SAMP8 suffers from increased oxidative stress and that accelerated senescence was associated with increased oxygen radicals synthesis. We have also recently demostrated that the anticontractile effect of perivascular fat is impaired in a senescence-accelerated prone mouse, compared with control senescence-resistant mice (SAMR1). A long-term treatment with melatonin seems to decrease contractile responses to norepinephrine in mesenteric small arteries of SAMP8, thus restoring an anticontractile effect, probably through antioxidant mechanisms. However, it is not know whether melatonin is able to modulate the production of adiponectin and/or the expression of adiponectin receptors. Therefore, the aim of the study was to investigate the production of adiponectin and/or the expression of adiponectin receptors in the visceral adipose tissue of SAMP8 before and after chronic treatment with melatonin. Design and method: We investigated 7 SAMP8 and 7 SAMR1 treated for 10 months with melatonin (MEL, 10 mg/kg/day), an endogenous indoleamine with antioxidant and vasculoprotective properties, as well as 7 SAMP8 and 7 SAMR1 untreated controls (CTR) kept untreated for 10 months. Visceral fat (perirenal fat) was obtained by dissection, and the adiponectin content, as well as the expression of adiponectin receptor were evaluated by immunohistochemistry. Results: Results are shown in the figure (* p < 0.05 vs SAMR1 CTR; # p < 0.05 vs SAMR1 + MEL; + p < 0.05 vs SAMP8 + MEL). The adiponectin content, as well as the expression of adiponectin receptor were reduced in untreated SAMP8 compared with untreated SAMR1. Treament with MEL was able to increase the production of both proteins in SAMP8. Figure. No caption available. Conclusions: Melatonin treatment is able to increase the production of adiponectin and the expression of adiponectin receptor in the visceral fat of aging mice. This observation might contribute to explain the improvement of the anticontractile action of perivascular fat observed in the mesenteric small resistance arteries of SAMP8 mice after chronic treatment with MEL.

CEREBRAL SMALL RESISTANCE ARTERY STRUCTURE AND CEREBRAL BLOOD FLOW IN NORMOTENSIVE SUBJECTS AND IN HYPERTENSIVE PATIENTS: 3D.01

We have previously demonstrated that, in essential hypertensive patients, subcutaneous small resistance artery structural alterations, as indicated by an increased media to lumen ratio (M/L), may predict coronary (J Hypertens 2003;21:625–631) and forearm (J Hypertens 1995;13:341–347) flow reserve. In essential hypertension also human cerebral small arteries present a clear increase in M/L (Rizzoni D et al, J Hypertens 2009; 27:838–845). However, there is no available demonstration about the presence of a direct relationship between cerebral blood flow and cerebral small resistance artery structure. Patients and Methods: Ten subjects were included in the present study. They were 5 hypertensive patients (HP) and 5 normotensive control subjects (NS). All subjects underwent a neurosurgical intervention (trauma, meningioma, etc.). A small portion of morphologically normal cerebral tissue was excised and rapidly put in chilled physiological saline solution. Cerebral small resistance arteries (relaxed diameter around 200 μm) were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured (M/L). Before surgical intervention we have also investigated cerebral blood flow (CBF) in different areas (cortex, lenticular nucleus, thalamus and subcortical white matter) by means of dynamic susceptibility contrast (DSC) MR imaging with bolus injection of gadolinium, focusing on specific regions of interest. Results: Are summarized in the Table (*=p < 0.05; ***p < 0.001 vs. NS). Figure 1. No caption available. A statistically significant inverse correlation was observed between M/L of cerebral arteries and CBF in the lenticular nucleus (r=−0.80, p < 0.01) and with CBF in the thalamus (r = −0.75, p < 0.01), while correlation with CBF in the cortex and in the subcortical white matter were not statistically significant (r = −0.21 and −0.21, respectively). Conclusions: Our results indicate that cerebral small artery structure might play a major role in controlling CBF, and this might help to explain the relevant role of structural alterations of small resistance arteries in predicting cerebrovascular events (Circulation 2003;108:2230–2235).

FIBRONECTIN AND LAMININ CONTENT IN THE TUNICA MEDIA OF SUBCUTANEOUS SMALL RESISTANCE ARTERIES OF PATIENTS WITH ESSENTIAL HYPERTENSION: PP.11.428

Objective: We have previously demonstrated that a pronounced fibrosis, due to an increased content in type 1 collagen, may be observed both in the tunica media and in the adventitia of subcutaneous small resistance arteries of patients with essential hypertension and with primary aldosteronism (JCEM 2006, 91:2638–2642). However, it was suggested that, in the development of hypertensive microvascular remodeling, a relevant role may be played by laminin and fibronectin vascular content, at least in animal models of hypertension. Aim of this study was to evaluate the amount of fibronectin and laminin within the tunica media of subcutaneous small arteries of normotensive subjects and essential hypertensive patients. Design and Methods: We have investigated 6 normotensive control subjects and 10 essential hypertensive patients. All subjects were submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, the fibronectin and laminin content within the tunica media was evaluated by immunohistochemistry, with subsequent image analysis (% of area stained). Results: As expected, clinic blood pressure values were higher in essential hypertensive patients (153/89 ± 7/11 mm Hg) than in normotensive controls (116/81 ± 10/5 mm Hg, P < 0.01). The media to lumen ratio was significantly increased in essential hypertensive patients (0.100 ± 0.03) compared with normotensive controls (0.06 ± 0.02, p < 0.01). Fibronectin media content was significantly greater in essential hypertensive patients (7.41 ± 2.28 %), compared with normotensive controls (5.62 ± 0.40, P < 0.05). A significant correlation was observed between fibronectin media content and media to lumen ratio (r = 0.49, p < 0.05). No significant difference in laminin media content was observed between groups (3.7 ± 1.71 % in essential hypertensive patients, 5.63 ± 1.79 % in normotensive controls). Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, fibronectin, but not laminin media content is increased. Fibronectin might be therefore involved in the development of small resistance artery remodeling in humans.

POOR CONCORDANCE BETWEEN NON-INVASIVE AND LOCALLY INVASIVE TECHNIQUES OF EVALUATION OF MICROVASCULAR MORPHOLOGY IN THE DETECTION OF HYPERTROPHIC REMODELING OF SMALL RESISTANCE ARTERIES

Objective: The evaluation of the morphological characteristics of small resistance arteries in human beings is not easy. The gold standard is generally considered to be the evaluation of the media to lumen ratio (MLR) of subcutaneous small vessels obtained by local biopsies and evaluated by wire or pressure micromyiography. However, non-invasive techniques for the evaluation of retinal arterioles were recently proposed, in particular two approaches seem to provide interesting information: scanning laser Doppler flowmetry (SLDF) and adaptive optics (AO); both of them provide an estimation of the wall to lumen ratio (WLR) of retinal arterioles, as well as of internal and external diameters and wall cross-sectional area. Design and method: In the present study we enrolled 12 normotensive subjects, 12 lean hypertensive patients (HP), 9 severely obese normotensive patients (ONP) and 8 severely obese hypertensive patients (OHP), undergoing an election surgical intervention. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance artery structure was assessed by wire micromyography and the MLR was calculated. WLR of retinal arterioles was obtained by Scanning Laser Doppler Flowmetry and AO (SLDF, Heidelberg Engineering, Heidelberg, Germany and RTX-1, Imagine Eyes, Orsay, France). In hypertensive/obese patients the characteristics of microvascular remodeling were assessed by the calculation of the remodeling and growth indices, according to Heagerty AM et al, Hypertension 1993; 21:391–391). The remodeling index quantifies the proportion of the increase in the MLR or WLR that may be explained by a re-arrangement of the same amount of wall material around a narrowed lumen (eutrophic remodeling), while the growth index quantifies the contribution of vascular smooth muscle cell hypertrophy or hyperplasia (hypertrophic remodeling).When eutrophic remodeling is present, the remodeling index is close to 100%. Results: are reported in the Table. Figure. No caption available. Conclusions: Our data suggest that the three methodological approaches seems are not completely concordant in the detection of hypertrophic remodeling, since data provided are in relative poor agreement. The presence of a relevant proportion of hypertrophic remodeling in obese patients (Hypertension 2011; 58:29–36), observed with micromyographic approaches, is not always confirmed by non-invasive approaches.

REGRESSION OF RETINAL MICROVASCULAR ALTERATIONS IN PATIENTS WITH RHEUMATOID ARTHRITIS TREATED WITH ABATACEPT, AN INHIBITOR OF LYMPHOCYTIC CO-STIMULATION

Objective: It has been previously demonstrated that T lymphocytes may be involved in the development of hypertension and microvascular remodeling, and that circulating T effector lymphocytes may be increased in hypertension (De Ciuceis C et al, Am J Hypertens 2017, 30:51–60; Itani HA et al. Hypertension 2016; 68:123–132). In particular, Th1 and Th 17 lymphocytes may contribute to the progression of hypertension and microvascular damage. Abatacept is a fusion protein composed of the Fc region of the immunoglobulin IgG1 fused to the extracellular domain of CTLA-4 that binds to the CD80 and CD86 molecule, and prevents activation of T cells, avoiding CD28 repetitive engagement. Abatacept is presently indicated for treatment of moderate to severe active rheumatoid arthritis in adults (aged 18 years or over) who have not had an adequate response to other drugs. The aim of our study was to non-invasively investigated morphological characteristics of retinal arterioles in patients with rheumatoid arthritis before and after treatment with abatacept. Design and method: In the present study we enrolled 5 patients with rheumatoid arthritis. The wall to lumen ratio (WLR) of retinal arterioles was measured by adaptive optics (RTX-1, Imagine Eyes, Orsay, France), a recently validated technique (De Ciuceis C, in press in J Hypertens) for non-invasive assessment of retinal arteriolar morphology, providing also an estimation of internal and external diameters and wall cross-sectional area. Morphological evaluations were performed at baseline, after 6 months and after 12 months of treatment Results: The results obtained are reported in the Table (* < 0.05, **p < 0.01 vs. baseline). Figure. No caption available. Conclusions: Our data suggest that a reduction in WLR, wall thickness and wall cross-sectional area was observed after treatment for 12 months with abatacept, thus suggesting the possibility to induce a regression of microvascular abnormalities through a modulation of the immune system.

[PP.09.17] REPRODUCIBILITY OF THE EVALUATION OF THE WALL TO LUMEN RATIO OF RETINAL ARTERIOLES WITH TWO DIFFERENT NON-INVASIVE APPROACHES: SCANNING LASER-DOPPLER FLOWMETRY AND ADAPTIVE OPTICS

Objective: The evaluation of the morphological characteristics of small resistance arteries in human beings in not easy. The gold standard is generally considered to be the evaluation of the media to lumen ratio of subcutaneous small vessels obtained by local biopsies and measured by wire or pressure micromyiography. However, non-invasive techniques for the evaluation of retinal arterioles were recently proposed, in particular two approaches seem to provide interesting information: scanning laser Doppler flowmetry and adaptive optics; both of them provide an estimation of the wall to lumen ratio (WLR) of retinal arterioles. The reproducibility of such measurements was previously stated to be acceptable (coefficient of variation <10% for SLDF, <4% for RTX-1), however, no direct comparison of the two techniques in the same population was previously performed. Design and method: Therefore, we evaluated 18 subjects and patients (10 normotensives, 8 hypertensives, 7/18 severely obese). In all of them an evaluation of the WLR of retinal arterioles was made by Scanning Laser Doppler Flowmetry (SLDF, Heidelberg Engineering, Heidelberg, Germany) and adaptive optics (RTX-1, Imagine Eyes, Orsay, France). The same operator evaluated the same acquired images in two different days (intra-observer variability), and two different operators evaluated the same images in the same day (inter-observer variability). Results: The results are reported in the Table (***p < 0.001). Variation coefficient of SLDF is much greater than that of AO. Figure. No caption available. Conclusions: It is clear how the reproducibility of the evaluation of the WLR with adaptive optics is far better, as compared with SLDF, since the variation coefficient are clearly lower. This may be important in terms of clinical evaluation of microvascular morphology in a clinical setting.

[OP.8C.03] COMPARISON BETWEEN THREE NON-INVASIVE TECHNIQUES OF EVALUATION MICROVASCULAR MORPHOLOGY VS. THE GOLD-STANDARD LOCALLY INVASIVE MICROMYOGRAPHY. PRELIMINARY DATA

Objective: The evaluation of the morphological characteristics of small resistance arteries in human beings in not easy. The gold standard is generally considered to be the evaluation of the media to lumen ratio (M/L) of subcutaneous small vessels obtained by local biopsies and evaluated by wire or pressure micromyiography. However, non-invasive techniques for the evaluation of retinal arterioles were recently proposed, in particular two approaches seem to provide interesting information: scanning laser Doppler flowmetry (SLDF) and adaptive optics (AO); both of them provide an estimation of the wall to lumen ratio (WLR) of retinal arterioles. A non-invasive measurement of basal and total capillary density may be obtained by videomicroscopy/capillaroscopy. No direct comparison of the three non-invasive techniques in the same population was previously performed, in particular AO was never validated against micromyography. Design and method: In the present study we enrolled 12 normotensive subjects and 8 hypertensive patients undergoing an election surgical intervention; 11/20 were severely obese). All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance artery structure was assessed by wire myography and the M/L was calculated. WLR of retinal arterioles was obtained by Scanning Laser Doppler Flowmetry and AO (SLDF, Heidelberg Engineering, Heidelberg, Germany and RTX-1, Imagine Eyes, Orsay, France). Functional (basal) and structural (total) microvascular density were evaluated by capillaroscopy (Videocap 3, DS Medica, Milan, Italy) before and after venous congestion. Results: The results are summarized in the Table (slope of the relation: p < 0.01 RTX-1 vs. SLDF). Figure. No caption available. Conclusions: Our data suggest that AO has a substantial advantage over SLDF in terms of evaluation of microvascular morphology, since it is more closely correlated with the M/L of subcutaneous small arteries, considered a gold-standard approach but limited in its clinical application by the local invasiveness of the procedure.

RELATIONSHIP BETWEEN MICROVASCULAR T REGULATORY LYMPHOCYTES AND CIRCULATING LYMPHOCYTES

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-&ggr;), Th 17 (producing IL-17) 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. 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). Design and method: Therefore, we evaluated the possible relationship between Treg detected in small resistance arteries and circulating levels of Treg. We enrolled 11 normotensive subjects and 4 hypertensive patients undergoing an election surgical intervention (usually removal of adrenal gland for a non-producing adenoma). No sign of local or systemic inflammation was present in any subjects or patients. All patients underwent a biopsy of subcutaneous fat during surgery and small resistance arteries were isolated. 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 assessment of T lymphocyte subpopulations. Lymphocyte phenotype was evaluated by flow cytometry after 5 days in vitro activation in order to assess Th17 lymphocytes. Results: A significant positive correlation was detected between Treg in small resistance arteries and circulating levels of Treg (R = 0.42, p∼0.05) whereas an inverse correlation was observed between Th17 lymphocytes and Treg in small resistance arteries (R = -0.46, p < 0.05). Conclusions: Our data suggest that Treg lymphocytes detected in subcutaneous small resistance artery wall are related to circulating Treg which were previously observed to inversely correlate with subcutaneous small resistance artery media to lumen ratio and retinal arteriole wall to lumen ratio. This suggest that Treg may be protective against microvascular damage confirming an involvement of adaptive immune system on microvascular remodeling.

INCREASED INTERLEUKIN-18 PLASMA LEVELS IN HYPERTENSIVE PATIENTS AFTER ORAL GLUCOSE TOLERANCE TEST: 8B.07

Type 2 diabetes mellitus is associated with an increased oxidative stress and/or inflammation, which might play a relevant role in the development of clinical complications. The aim of the present study was to investigate circulating indices of inflammation/oxidative stress during an oral glucose tolerance test (OGTT) in hypertensive patients. We have enrolled 14 essential hypertensive patients with at least two components of the metabolic syndrome, but without overt diabetes mellitus. All patients underwent a standard OGTT (75 mg glucose). Interleukin 6, interleukin 18, lipoperoxydes, antioxidant capacity (PAO) and macrophages chemotactic factor 1 (MCP-1) plasma circulating levels were evaluated before and 120’ after OGTT by ELISA. Results: OGTT demonstrated an impaired glucose tolerance in all cases (basal: 102 ± 11 mg/dl, 120’ 221 ± 24 mg/dl). Plasma interleukin 18 was significantly increased after OGTT (2069 ± 1010 vs. 1305 ± 546 pg/ml, p = 0.02). No increase was observed in plasma interleukin 6 (6.6 ± 5.38 vs. 8.02 ± 7.91 pg/ml p = NS), plasma lipoperoxydes 5.39 ± 6.89 vs. 4.87 ± 4.97 μM/L, p = NS), PAO (630 ± 137 vs. 688 ± 180 μM/L, p = NS) and plasma MCP-1 (527 ± 450 vs. 376 ± 290 pg/ml, p = NS). Blood pressure values were 145 ± 91 ± 11/9 mm Hg; body mass index 27 ± 3 Kg/m2. In conclusion, our data suggest that an increase in serum glucose during OGTT is associated with systemic inflammation, as documented by the observation of an increase in plasma interleukin 18, but not with an increased oxidative stress.