Abstracts
phosphatidylserine concentration and the number and dimension of
MP were assessed by nanoparticle tracking analysis (NTA). Using
a Nanosight LM10-HS system (NanoSight Ltd., Amesbury, UK), MP
were visualized by laser light scattering. MP-associated TF activity
was assessed by a one-stage clotting assay. To assess intracellular
calcium mobilization, HMC and HUVEC were loaded with Fluo-4-NW
probe and analyzed with a fluorimeter. All data are expressed as
mean ± SEM.
Results: PM treatment induced a dose-dependent, rapid increase in
MP generation; at 1 h and 500 μg/mL, MP-associated phosphatidylserine
concentration went from 0.09 ± 0.02 nM to 0.37 ± 0.13 nM for HMC
(p b 0.05) and from 7.13 ± 0.51 nM to 24.06 ± 6.98 nM (p b 0.001)
for HUVEC. The results were confirmed by Nanosight analysis under
the same conditions (HMC: 0.32 ± 0.02.106 events/mL and 0.86 ±
1.10.106 events/mL for unstimulated vs. PM-stimulated cells, respective-
ly; HUVEC: 1.28 ± 0.08.106 events/mL and 2.32 ± 0.15.106 events/mL
for unstimulated vs. PM-stimulated cells; p b 0.0001 for both compari-
sons). PM treatment induced an increase in MP-associated TF activity
(HMC: 6.33 ± 0.82 mU and 13.17 ± 0.49 mU for unstimulated vs. PM-
stimulated cells; HUVEC: 12.42 ± 4.78 mU and 35.65 ± 10.86 mU for
unstimulated vs. PM-stimulated cells; p b 0.001 for both comparisons).
Finally, PM treatment caused intracellular Ca++ mobilization in both
cells types.
Conclusions: PM-mediated generation of procoagulant, TF-
bearing MP by HMC and HUVEC might contribute to the development
of cardiovascular diseases upon exposure to airborne pollutants.
doi:10.1016/j.vph.2015.11.059
Endothelial cells stimulate differentiation of circulating
endothelial precursors through soluble factors
S. Paccosia,, G. Grazianib, R. Caporalec, A.M.G. Gellic, A. Parentia
a
Department of Health Sciences, Clinical Pharmacology and Oncology
Section, University of Florence, Florence, Italy
b
Blood Transfusion Centre, Italy
c
Central Laboratory, Azienda Ospedaliero-Universitaria Careggi,
Florence, Italy
Objectives: Several studies have demonstrated that endothelial
progenitor cells (EPCs) from either the bone marrow or circulating
blood, are essential for functional recovery of ischemic tissue after
cell therapy. Although promising preliminary results, the mecha-
nisms by which EPCs interact with vascular wall cells and ischemic
tissues remain unclear. We have previously reported that human
coronary artery endothelial cells (HCAECs) co-cultured with periph-
eral blood mononuclear cells (PBMCs) can stimulate their early
differentiation toward a pre-endothelial phenotype. We investigated
possible soluble factors, released from the co-culture, involved in EPC
differentiation.
Materials and methods: PBMCs isolated from healthy donors
were co-cultured with human coronary endothelial cells (HCAECs)
by means of 0.4 μm pore sized membranes transwell. EPC differen-
tiation was evaluated by means of flow cytometry. Milliplex assay
was used to measure soluble factors.
Results: Ten factors released by co-cultures were measured and
among these, IL-6, IL-8, EGF and CCL-2 levels were significantly
higher than that found in HCAECs or in PBMCs alone. In order to
check their involvement in PBMC differentiation, blocking experi-
ments with neutralizing antibodies were performed. Flow cytometry
analysis confirmed an impairment of PBMC differentiation toward
a pre-endothelial phenotype when IL-6, IL-8 and with a lesser extent
CCL-2 were blocked.
Conclusions: These data add a new insight into the mechanisms
by which endothelial precursors interact with vascular wall, thus
65
suggesting future directions in understanding and treating ischemic
injury.
doi:10.1016/j.vph.2015.11.060
Dendritic cell phenotype and function are modulated by
inflammation and insulin-resistance
A. Parentia,, S. Paccosia, L. Palab, A. Silvanoc, V. Barbarob,
C.M. Rotellab,d, P. Romagnolic
a
Department of Health Sciences, Clinical Pharmacology and Oncology
Section, Italy
b
Section of Endocrinology, Careggi University Hospital, Florence, Italy
c
Department of Experimental and Clinical Medicine, Italy
d
Department of Biomedical, Experimental and Clinical Sciences,
University of Florence, Florence, Italy
Objectives: Inflammation is involved vascular remodeling. Den-
dritic cells (DCs) are antigen presenting cells which may localize in
the healthy human arterial wall and increase in the atherosclerotic
lesion. Obesity and insulin resistance are well known risk factors for
cardiovascular disease, and diabetic patients with atherosclerotic
disease have compromised immune functions. We have previously
reported that myeloid precursors of DCs isolated from obese and
diabetic patients (obese T2D) show quantitative abnormalities and
significantly increase their adhesiveness to vascular coronary smooth
muscle cells. We then further in vitro characterized DC obtained in
vitro from obese T2D patients.
Materials and methods: DCs were obtained from circulating
PBMC of obese and obese T2D patients and characterized by flow
cytometry. Expression of integrins mRNAs was assessed by real time
PCR. Activation and proliferation of lymphocytes were performed by
CFSE assay.
Results: Real time PCR demonstrated that DCs from obese
T2D patients significantly increased the expression of CD18, CD11c
and DC-SIGN, while no differences were found in obese patients
compared to control subjects. CFSE analysis demonstrated that T2D
DCs were less able to stimulate lymphocyte proliferation, in line with
the phenotypical characterization, compared to control subjects. No
morphological differences were found between T2D DCs and healthy
controls, by means of electron microscopy.
Conclusions: Inflammation and insulin-resistance affect DC
phenotype and function. Therefore DCs may be candidate to a major
role in the onset and progression of vascular inflammation and
remodeling, suggesting that they could be a target for therapy.
doi:10.1016/j.vph.2015.11.061
Exacerbation of myocardial ischemia/reperfusion injury induced
by high-fat-high-fructose (HFHF) diet: Role of NLRP3 inflammasome
F. Tullioa,, F. Chiazzab, R. Mastrocolaa, C. Pennaa, D. Nigroa, V. Fracassoa,
G. Alloattic, R. Fantozzib, M. Aragnoa, M. Collinob, P. Pagliaroa
a
Department of Clinical and Biological Sciences, Italy
b
Department of Drug Science and Technology, Italy
c
Department of Life Sciences and Systems Biology, University of Turin,
Turin, Italy
Objectives: The diet-induced metabolic overload initiates a low-
grade, chronic inflammatory response, known as metaflammation,
which promotes cardiometabolic diseases. A recently identified
pathways involved in metaflammation is the NLRP3 inflammasome,
a large multimeric danger-sensing platform that promotes autocat-
alytic activation of the cysteine protease caspase-1 and mediates the