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Objective: The gold standard technique of evaluation microvascular morphology in human is generally considered the measure of media to lumen ratio (M/L) of subcutaneous small vessels obtained by local biopsies and evaluated by wire or pressure micromyography. 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 non-invasive techniques in the same population was previously performed, in particular AO was never validated against micromyography. Methods: 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 SLDF 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 before and after venous congestion. Results: The results are summarized in the Table (slope of the relation: p < 0.01 RTX-1 vs. SLDF). Basal capillary density in the nailfold/M/L Total capillary density in the forearm / ML Basal capillary density in the dorsum of the finger / M/L Total capillary density in the dorsum of the finger /ML Correlation coefficients (n = 20) 0.53, r2 = 0.28, p < 0.05 0.50, r2 = 0.25, p < 0.05 0.17, r2 = 0.29, p = NS 0.34, r2 = 0.12, p = NS W/L retinal arterioles (SLDF)/ M/L W/L retinal arterioles (RTX- 1)/M/L W/L retinal arterioles (SLDF)/ W/L retinal arterioles (RTX-1) Correlation coefficients (n = 20) 0.54, r2 = 0.29, p < 0.01 0.90, r2 = 0.81, p < 0.001 0.71, r2 = 0.50, p < 0.001 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.

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. In particular, Th1 and Th17 lymphocytes may contribute to the progression of hypertension and microvascular damage while TREG lymphocytes seem to be protective. However, no data is available about patients with severe obesity, in which pronounced microvascular alterations were observed. Methods: We have investigated 32 severely obese patients undergoing bariatric surgery, 24 normotensive lean subjects and 11 hypertensive lean subjects undergoing an election surgical intervention. No sign of local or systemic inflammation was present in any subject or patient. A peripheral blood sample was obtained before surgery for assessment of CD4+ T lymphocyte subpopulations. Lymphocyte phenotype was evaluated by flow cytometry after 5 hour in vitro activation in order to assess T-effector and T-regulatory (TREG) lymphocytes. Subsets of TREGS were defined as follows: −TREGS recent thymic emigrants (RTE), directly derived from thymus: CD31+; −TREGS naïve: CCR7+CD45RA+; −TREGS central memory (CM): CCR7+CD45RA−; −TREGS effector memory (EM): CCR7−CD45RA−; −TREGS terminal differentiated effector memory (TDEM): CCR7−CD45RA+. Results: The results are summarized in the Table (*p < 0.05, **p < 0.01, ***p < 0.001 vs. lean normotensives; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. lean hypertensives). A marked reduction of several TREG subpopulations was observed in obese patients compared with controls, together with an increased in some T-effector cells. Lean normotensives Lean hypertensives Obese patients TREGs (%) 4.11 ± 1.60 4.64 ± 1.66 2.69 ± 1.81**## TREGs (abs number) 45.4 ± 24.3 45.4 ± 23.8 27.3 ± 21.1**# TREGs naíve (%) 22.1 ± 10.1 18.1 ± 13.1 13.34 ± 12.9** TREGs naíve (abs number) 10.6 ± 7.75 9.71 ± 8.87 3.87 ± 5.28***## TREG CM (%) 32.3 ± 13.8 32.8 ± 17.8 22.7 ± 15.2*# TREGs CM (abs number) 14.7 ± 10.2 14.2 ± 9.08 6.10 ± 8.08***## CD4+ EM (%) 24.4 ± 9.96 26.8 ± 12.5 34.1 ± 13.3** CD161+CD28+ (%) 86.2 ± 28.5 94.9 ± 5.63 97.2 ± 5.39* Conclusion: TREG lymphocytes are clearly reduced in severely obese patients, possibly contributing to the development of marked microvascular alterations previously observed in such a population.

Background. The anticontractile effect of perivascular adipose tissue(PVAT) is lost in obese patients due to adipocyte hypertrophy, leading to hypoxia, inflammation, and oxidative stress(Circulation 2009; 119(12):1661–1670).We recently demonstrated that the property of PVAT, partially maintained in an animal model of genetic obesity, seems to be related to the activity of BKCA channels, since is selectively blocked by iberiotoxin. We aimed to investigate functional responses of small mesenteric arteries in an animal model of genetic obesity after chronic treatment with melatonin, an endogenous hormone with antioxidant and vasculoprotective properties. Methods. Obese mice (n = 9) (B6.V-Lep ob/OlaHsd, Harlan Laboratories S.r.l.) (ob/ob) and control lean mice (n = 8) (CLM) were treated with melatonin (MEL)100 mg/kg per day for 8 weeks (from the5th to the 13th week of age).Data were compared from untreated ob/ob (n = 15) and CLM (n = 10) animals. Mesenteric small resistance arteries were dissected and mounted on a wire myograph. Concentration-response to norepinephrine was evaluated in vessels with intact PVAT (WF) and in vessels in which PVAT was removed (NoF) under normoxic and hypoxic (30’95% N2/5%CO2) conditions. Norepinephrine concentration-response curve was repeated with iberiotoxin(30’preincubation 100 nm/L). Results. MEL significantly reduced the contractile response in NoF ob/ob and CLM vessels (ANOVA P = 0.014 and P = 0.049 respectively).The improvement after MEL was also seen in CLM NoF vessels during hypoxia(ANOVA p < 0.05) and following preincubation with iberiotoxin(P < 0.05), with no significant improvements in ob/ob. Increases in contractility following hypoxia and iberiotoxin treatment were restored byMEL in Ob/Ob WF vessels (P = 0.013 and P = 0.036 respectively),whereas MEL only rescued the effects of hypoxia(P = 0.045) in CLM WF arteries. In conclusion, MEL exerts a protective effect in small vessels with and without PVAT from both ob/ob and CLM, counteracting the adverse effect of hypoxia and iberiotoxin in vessels with PVAT and in CLM vessels whitout PVAT. However, in Ob/Ob animals MEL rescues the effects only in the presence of PVAT indicating the importance of PVAT oxidative stress in vascular dysfunction observed in Ob/Ob animals.

Objective: Wall-to-lumen ratio of retinal arterioles might serve as an in-vivo parameter of vascular damage. Previous studies have shown a correlation between retinal arteriolar structure and blood pressure values, measured both in the clinic and during 24 hours ambulatory monitoring. We analyzed the impact of brachial clinic blood pressure (BP), of central BP and of 24 hours BP values on wall-to-lumen ratio of retinal arterioles. Methods: In 267 subjects (129 males, age range 20–72 years; mean 54±7 years) wall-to-lumen ratio of retinal arterioles was assessed in vivo using scanning laser doppler flowmetry (Heidelberg retina flowmeter, Heidelberg Engineering). In addition clinic and 24 hours BP values were measured. Central hemodynamics and augmentation index (Ai) were assessed by pulse wave analysis. Results: In never treated patients with essential hypertension (n=56) a higher wall-to-lumen ratio (0.37±0.19 vs. 0.30±0.13, P=0.05) was observed in comparison with normotensive individuals (n=115); no significant differences were observed between treated (n=96) and untreated hypertensive patients. Wall to lumen ratio and wall cross sectional area of retinal arterioles were significantly related to clinic systolic BP(r=0.23, P=0.005) and PP(r=0.18, P=0.005), to 24 hours systolic BP(r=0.28, P=0.0001) and PP(r=0.19, P=0.003) and to central systolic BP(r=0.20, P=0.01) and central PP(r=0.21, P=0.001). Multiple regression analysis including all BP indices revealed that only mean systolic 24 hours BP is independently associated with an increased wall-to-lumen ratio of retinal arterioles. Conclusion: in this quite large group of hypertensive patients and normotensive individuals 24 hours systolic BP seems to be the strongest determinant of increased WLR of retinal arterioles.