SUPPLEMENTAL ONLINE MATERIAL

MATERIALS AND METHODS SECTION:

Evaluation of circulating and vascular T lymphocyte subpopulations

Evaluation of circulating CD4+ T lymphocyte phenotype

Peripheral blood CD4+ T-cell phenotypic characterization was performed by flow cytometry

(Cytomics NAVIOS, Beckman Coulter Inc., Fullerton, CA), as previously reported (1): 100

microliters of whole blood were incubated mixture of monoclonal antibodies (from Beckman

Coulter, R&D Systems, Caltag Laboratories, Becton Dickinson, San Jose, CA), conjugated with

fluorochromes at 4°C for 30 minutes. Double-negative CD3+CD4-CD8- T-cells were evaluated in

all patients and no difference between normotensive and hypertensive individuals was observed.

Absolute cell count was determined by single-platform analysis using Flow-Count beads (Beckman

Coulter).

CD4+ TREG were defined by the high expression of CD25 and the low expression/absence of

CD127, using PC5-conjugated anti-CD4, PE-conjugated anti-CD25 and PC-7 conjugated anti-

CD127 (2) In addition, TREG were evaluated with FITC-conjugated anti-CD31 and ECD-

conjugated CD45RA (Beckman Coulter), in order to divide them into a subset directly derived from

the thymus (CD45RA+CD31+) and a subset induced in periphery (CD45RA neg).

In addition, different subpopulations of TREGS were identified using, in two separate tubes, FITC-

conjugated CCR7 or FITC-conjugated CD31 and ECD-conjugated CD45RA.

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+.

Examples of flow cytometry data and the gating strategy used are reported in the Figure.1

Cytokine production by CD4 + T cells after in vitro activation.

Peripheral blood mononuclear cells obtained by Ficoll-Hypaque gradient centrifugation were cul-

tured at 37°C temperature and 5% CO2 pressure, and stimulated with phorbol 12-myristate 13-ac-

etate (PMA, 5 ng/ml; Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) and ionomycin (500

ng/ml; Sigma-Aldrich), for 5 hours in the presence of monensin (1µM; Sigma-Aldrich), with or

without 5 days of pre-activation with anti-CD3 (20 UI/ml; Sigma Aldrich) and anti-IL2 (0.2 µm;

Sigma-Aldrich). Since it is well-known that PMA induces a rapid down-regulation of CD4 mole-

cule (3) that prevents direct evaluation of the CD4+ populations, CD3+CD8- cells were considered

as CD4+ in the analysis of cytokine producing cells. Cells were washed and stained with PC5-con-

jugated anti-CD3, ECD-conjugated anti-CD28 and PC7-conjugated anti-CD8 for 30 minutes at

room temperature, fixed with paraformaldehyde (4%) for 5 minutes and permeabilized with saponin

(0.2%; Sigma-Aldrich) for 15 minutes. Intra-cytoplasmic staining was performed for 30 minutes us-

ing PE-conjugated anti-γ-IFN and Alexa Fluor 488-conjugated anti-IL-17A, or FITC-conjugated

anti-IL-5 and PE-conjugated anti-IL-4.

TREG lymphocyte evaluation in small resistance arteries by Methylation PCR Analysis

FoxP3 DNA was assessed from subcutaneous small resistance arteries by using The EpiTect Methyl II

PCR System kit, as previously described (4,5). The method is based on the detection of remaining input

DNA after cleavage with methylation-sensitive and methylation-dependent restriction enzymes. The

restriction digestions were performed by using the EpiTect Methyl II DNA Restriction Kit provided

by Qiagen (Chatsworth, CA) according to the manufacturer’s instructions. A reaction mix (RM)

without enzymes was prepared from 100 ng genomic DNA, 13l of 5x Restriction Digestion

Buffer, and RNase-DNase free water up to 60 l. Four separate digestions (Mo, Ms, Md, and Msd)

were prepared as follow: each one consists of 14l of the RM and 1l of RNase-DNase free water

(Mo digest), 1l of methylation sensitive enzyme A and 1l of RNase-DNase free water (Ms 2

digest), 1l methylation sensitive enzyme B and 1l of RNase-DNase free water (Md digest), 1l of

methylation sensitive enzyme A and 1l of methylation sensitive enzyme B (Msd digest). All 4

tubes were incubated over night at 37°C.

The digested DNA samples were then analyzed using the Human EpiTect Methyl II Signature PCR

Array for FoxP3 (EPHS115010-1A, Qiagen, Chatsworth, CA) according to the manufacturer’s

instructions. The Real-time PCR assay was performed in an ABI PRISM 7500 Fast Real Time

system (Applied Biosystems, Foster City, CA).

The data were analyzed using an integrated Excel-based template that automatically performs all

Ct based calculations from the raw threshold cycle (Ct) values to determine gene specific DNA

methylation status, using MethylScreenTM technology. The Excel template normalizes the Ct values

of both digests with the mock digestion values to calculate and report the percentage of the DNA

that is methylated and unmethylated. All experiments were performed in duplicates. FoxP3

unmethylated DNA fraction was considered specific for TREG lymphocyte population (4, 6) In fact,

methylation assay reliably reports Treg numbers in several diagnostic or therapeutic settings (6).

Evaluation of retinal arteriolar morphology

Wall to lumen ratio of retinal arterioles was assessed using Scanning Laser Doppler Flowmetry

(SLDF) at 670 nm (Heidelberg Retina Flowmeter, Heidelberg Engineering, Heidelberg, Germany),

an established and validated method to investigate the morphology of retinal arterioles (7-12).

Briefly, an arteriole with a size between 80 and 140 μm of the superficial retinal layer in a retinal

sample of 2.56 X 0.64 X 0.30 mm was scanned within 2 seconds, at a resolution of 256 points x 64

lines x 128 lines. Measurements were performed in the juxtapapillary area of the right eye, 2 to 3

mm temporal superior to the optic nerve; the mean from 3 measurements was taken (7-12). The

examination was performed without mydriasis, in sitting position after 20 minutes of rest, at room

temperature and daylight conditions between 8 AM and 2 PM, but before lunch. Analyses of

diameters were performed offline with automatic full-field perfusion imaging analysis program

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(Nirox Optoelectronics, Brescia, Italy). Outer arteriole diameter (AD) was measured in reflection

images, and lumen diameter (LD) was measured in perfusion images (10-12). Wall to lumen ratio

(W/L) was calculated using the formula (AD-LD)/LD (10-12). Reliability analysis of the wall to

lumen ratio pointed out a good reproducibility of the SLDF measurement, with an intraobserver

coefficient of variation around 8-9% and intraobserver coefficient of variation around 9-10% (9,12).

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Evaluation of skin capillary density

After a period of rest in sitting position in a quiet and temperature controlled room (21-22°C),

capillaries from nailfold and the dorsum of the fourth finger of the non-dominant hand were

visualized by using an epi-illuminated microscope containing a 100 W mercury vapour lamp light

source, and pictures (final magnification of 200) were obtained by video-microscopy (Videocap 3.0

D1 200, DS Medica, Milano, Italy) in baseline conditions (baseline capillary density) and after

venous congestion (total capillary density), in order to visualize functionally excluded capillaries.

Venous congestion was induced by inflating at to 60 mm Hg for 2 minutes a miniature blood

pressure cuff applied to the base of the fourth finger of the non-dominant hand (13-15). Images

(final magnification of 200) were also obtained before and after venous congestion at the distal third

forearm on the sagittal line by using a traditional pressure cuff. Capillary density was defined as the

number of capillaries per square millimeter of the microscopic field and was counted by hand. The

first row of the nailfold capillaries was considered. Capillary density was determined by two

independent operators and findings were averaged

RESULTS SECTION:

Hypertensive patients were older and had a calculated creatinine clearance (MDRD formula) lower

than normotensive controls. Serum creatinine was within normal limits in more than 90% of

subjects and patients. No difference between groups was present in indications to surgery.

Microvascular structure and circulating T- lymphocytes

No significant difference in morphological parameters of retinal arterioles was detected between

hypertensive patients and normotensive subjects (Table 1). Basal and total capillary density were

similar in the different locations explored: nailfold and dorsum of the 4th finger of the non-dominant

hand and anterior surface of the non-dominant forearm (Table 1). However, the difference between

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basal and total capillary density, both calculated as delta gain or % of capillary recruitment, was

smaller in hypertensive patients than in normotensive subjects (Table 1).

The percentage and absolute number of circulating CD4+CD28- T-cells producing IFN- γ after in

vitro activation were higher in hypertensive patients than in normotensive subjects (Table 1).

No other difference in circulating T-lymphocytes was observed between the two groups.

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TABLES

Table 1

Circulating CD4+ T-lymphocyte subpopulations, and structural characteristics of microvessels in

the examined population.

Total

population

(n=36)

Hypertensive

patients (n=12)

Normotensives subjects

(n=24)

Circulating lymphocytes

TREG (% of CD4+ T cells) 4.27±1.64 4.67±1.59 4.07±1.66

TREG (cell/µl) 43.7±24.6 44.1±25.9 43.6±24.6

TREG RTE (% of TREGs) 8.23±5.82 5.91±3.96 9.40±6.31

TREG RTE (cell/µl) 3.72±3.41 2.66±2.19 4.25±3.82

TREG naive (% of TREGs) 20.6±10.8 19.1±13.0 21.4±9,81

TREG naïve (cell/µl) 9.80±7.66 9.85±8.75 9.77±7.27

TREG CM (% of TREGs) 33.3±15.4 36.5±19.6 31.7±13.1

TREG CM (cell/µl) 14.1±9.02 14.8±8.50 13.7±9.45

TREG EM (% of TREGs) 41.9±17.5 41.3±23.3 42.3±14.4

TREG EM (cell/µl) 18.8±12.5 19.7±13.5 18.4±12.3

TREG TDEM (% of TREGs) 3.87±7.23 2.20±2.33 4.71±8.65

TREG TDEM (cell/µl) 1.49±2.73 0.94±1.06 1.77±3.25

TREG RTE (%) 20.8±8.22 18.1±7.61 22.2±8.34

TREG RTE (abs number) 215±126 177±112 234±130

Circulating lymphocytes (After 5

hours of activation)

Th1 IFNγ+ ( %) 7.14±4.2 8.05±3.16 6.69±4.57

Th1 IFNγ +( abs.number) 73.6±56.2 80.2±48.6 70.26±60.4

Th1 IFNγ+ CD28- (%) 0.84±1.29 1.65±1.87 * 0.47±0.70

Th1 IFNγ+ CD28- (abs. number) 7.24±11.52 15.2±18.2 ** 3.60±2.98

Th2 IL5+ (%) 5.57±4.80 5.73±5.21 5.49±4.71

Th2 IL5+ (abs. number) 60.2±64.9 59.0±58.1 60.8±69.4

Th2 IL5+CD28+ (%) 4.46±4.26 4.26±4.57 4.56±4.23

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Th2 IL5+CD28+ (abs. number) 47.9±57.4 40.7±49.9 51.5±61.5

After 5 days of activation

Th2 IL4+ (%) 3.69±2.88 4.36±3.64 3.78±3.09

Th2 IL4+ (abs number) 35.0±33.2 37.9±38.5 37.9±36.6

Th2 IL4+ CD28+ (%) 2.90±2.52 3.51±2.61 2.82±2.60

Th2 IL4+ CD28+ (abs number) 26.5±27.7 27.6±27.7 28.0±29.4

Th2 IL5+ (%) 8.08±10.7 7.66±9.77 8.44±11.0

Th2 IL5+ (abs number) 71.2±104 58.2±61.8 80.1±119

Th2 IL5+ CD28+ (%) 6.77±9.67 6.86±9.47 6.66±9.75

Th2 IL5+ CD28+ (abs number) 56.2±87.2 44.2±49.9 62.1±99.6

Th2 IL5+ CD28- (%) 1.32±1.56 1.5±1.99 1.49±1.76

Th2 IL5+ CD28- (abs number) 11.8±20.1 12.8±21.1 14.0±23.1

Th17 IL17+ (%) 5.32±5.99 4.32±4.77 5.64±6.46

Th17 IL17+ (abs number) 53.0±68.3 42.4±54.6 56.8±73.8

Retinal arterioles

External diameter (µm) 99.3±19.9 93.4±25.1 102±16.9

Internal diameter (µm) 75,6±15.9 70.7±18.8 77.9±14.3

Wall thickness (µm) 11.9±5.09 11.3±4.58 12.2±5.42

Wall cross sectional area (µm2) 3408±1885 3115±2017 3547±1859

Wall to lumen ratio 0.33±0.14 0.33±0.12 0.33±0.16

Capillary morphology (nailfold)

Basal capillary density 8,20±1.88 8.25±0.96 8.19±2.07

Total capillary density 8.30±1.80 7.20±2.49 8.31±1.99

Delta gain 0.10±0.45 0.27±0.90 0.13±0.5

% capillary recruitment 1.25±5.59 0.00±0 1.56±6.25

Capillary morphology (dorsum of

the 4th left finger)

Basal capillary density 80.7±16.6 85.0±13.3 78.7±17.8

Total capillary density 93.1±16.5 88.6±15.7 95.1±16.8

Delta gain 12.4±12.5 3.56±4.77 ** 16.4±12.9

% capillary recruitment 12.9±13.7 3.63±4.50 * 17.1±14.4

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Capillary morphology

(forearm)

Basal capillary density 65.1±16.9 66.0±16.9 64.6±17.3

Total capillary density 73.8±22.2 78.2±21.9 71.6±22.6

Delta gain 8.68±9.67 12.2±10.6 7.00±9.01

% capillary recruitment 10.3±10.1 14.3±11.7 8.48±9.07

Abs. number: absolute number, TREG RTE: recent thymus emigrant cells; TREG Naïve: no

previous contact with antigens; TREG CM; T Central Memory cells; TREG EM: effector memory

cell (highly antigen-experienced cells active in adaptive response); TREG TDEM: TDEM Terminal

Differentiated Effector Memory: highly antigen-experienced cells that assume pro-apoptotic

properties, * P<0.05, ** P<0.01, vs. Normotensives subjects

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FIGURE 1

Flow cytometry evaluation (dot blot) in representative subjects of the two subgroups.

A. Dot plot analysis is of CD3+CD4+ gated lymphocytes. TREG were CD25high and CD127low.

B. Dot plot analysis is of CD3+CD8- gated lymphocytes after in vitro activation with PMA + Iono-

mycin. Th17 were IL-17+ among CD28+ or CD28- T cells.

C. Dot plot analysis is of CD3+CD8- gated lymphocytes after in vitro activation with PMA + Iono-

mycin. Th1 were IL- IFN-γ+ among CD28+ or CD28- T cells.

At least 20,000 events were analyzed for each sample. IFN-γ: interferon gamma; IL: interleukin.

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