Emanuela Mensà

MiR-21-5p and miR-126a-3p levels in plasma and circulating angiogenic cells: relationship with type 2 diabetes complications

Innovative biomarkers are required to manage type 2 diabetic patients (T2DM). We focused our study on miR-126-3p and miR-21-5p levels, as biomarkers of endothelial function and inflammation. MiRNAs levels were measured in plasma from 107 healthy subjects (CTR) and 193 diabetic patients (T2DM), 76 without (T2DM NC) and 117 with (T2DM C) complications. When diabetic complication were analysed as a whole, miR-126-3p and miR-21-5p levels declined significantly from CTR to T2DM NC and T2DM C patients. When miRNAs levels were related to specific complications, significantly higher miR-21-5p levels (0.46 ± 0.44 vs. 0.26±0.33, p < 0.001) and significant lower miR-126-3p levels (0.21±0.21 vs. 0.28±0.22, p = 0.032) were found in T2DM with previous major cardiovascular events (MACE) vs. all the others T2DM patients. To confirm these results we focused on circulating angiogenic cells (CACs) from a subgroup of 10 CTR, 15 T2DM NC and 15 T2DM patients with MACE. CACs from T2DM patients expressed higher miR-21-5p and lower miR-126-3p levels than CACs from CTR. Furthermore, CACs from T2DM + MACE showed the highest levels of miR-21-5p. Circulating miR-21-5p and miR-126-3p emerge as dynamic biomarkers of systemic inflammatory/angiogenic status. Their expression levels in CACs from T2DM with MACE suggest a shift from a proangiogenic to a proinflammatory profile.

HtrA1 in human urothelial bladder cancer: A secreted protein and a potential novel biomarker

Our aim was to analyze the expression of the serine protease HtrA1 in human bladder tissue and urine in order to point out its possible association with the presence of urothelial bladder cancer. Bladder tissue and urine specimens from cancer patients with different tumor grades and stages (n = 68) and from individuals with cystitis (n = 16) were collected along with biopsy specimens and urine from healthy individuals (n = 68). For the first time, we demonstrated by immunohistochemistry that HtrA1 protein is produced by bladder urothelium in both physiological and inflammatory conditions, whereas it is not detectable in urothelial cancer cells regardless of tumor grade and stage. A different HtrA1 expression between normal‐looking and neoplastic bladder tissue, despite similar HtrA1 mRNA levels, was also found by western blotting, which disclosed the presence of two forms of HtrA1, a native form of ∼50 kDa and an autocatalytic form of ∼38 kDa. Our investigations documented the presence of the two forms of HtrA1 also in urine. The ∼38 kDa form was significantly down‐regulated in neoplastic tissue, whereas significantly higher amounts of both HtrA1 forms were found in urine from cancer patients compared with both healthy subjects and patients with cystitis. Our findings suggest that HtrA1 is a downexpressed molecule since an early stage of bladder urothelial carcinoma development and that urinary HtrA1 protein may be considered, if successfully validated, as an early and highly sensitive and specific biomarker for this neoplasia (the sensitivity and specificity of HtrA1 are 92.65% and 95.59%, respectively).

Characterization of NO-producing neurons in the rat corpus callosum

The aim of this study was to determine the presence and distribution of nitric oxide (NO)‐producing neurons in the rat corpus callosum (cc).

Leukocyte telomere length and mortality risk in patients with type 2 diabetes.

Leukocyte telomere length (LTL) shortening is found in a number of age-related diseases, including type 2 diabetes (T2DM). In this study its possible association with mortality was analyzed in a sample of 568 T2DM patients (mean age 65.9 ± 9 years), who were followed for a median of 10.2 years (interquartile range 2.2). A number of demographic, laboratory and clinical parameters determined at baseline were evaluated as mortality risk factors. LTL was measured by quantitative real-time PCR and reported as T/S (telomere-to-single copy gene ratio). Age, gender, creatinine, diabetes duration at baseline, and LTL were significantly different between T2DM patients who were dead and alive at follow-up. In the Cox regression analysis adjusted for the confounding variables, shorter LTL, older age, and longer disease duration significantly increased the risk of all-cause mortality (HR = 3.45, 95%CI 1.02-12.5, p = 0.004). Kaplan-Maier analysis also found a different cumulative mortality risk for patients having an LTL shorter than the median (T/S ≤0.04) and disease duration longer than the median (>10 years) (log-rank = 11.02, p = 0.011). Time-dependent mortality risk stratification showed that T2DM duration and LTL combined was a fairly good predictor of mortality over the first 76 months of follow-up. In conclusion, LTL combined with clinical parameters can provide additive prognostic information on mortality risk in T2DM patients.

Physical activity and progenitor cell-mediated endothelial repair in chronic heart failure: Is there a role for epigenetics?

Chronic heart failure (CHF) is the most common cardiac disease among the elderly and a leading cause of mortality in elderly patients. Endothelial dysfunction is held to have a major role in the development and progression of CHF, which results in progressively impaired functional capacity. Endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs) are the main players involved in the endogenous repair mechanisms that can counteract endothelial dysfunction. A mounting body of data indicates that exercise enhances endothelial renewal through mobilization of bone marrow-derived EPCs and CACs, making it an effective therapeutic tool for CHF. Interestingly, emerging evidence has been showing that exercise training can also promote epigenetic modifications, e.g. DNA methylation, histone modifications, and differential expression of specific non-coding RNAs like microRNA (miRNAs). Since deregulation of the miRNAs involved in endothelial function modulation has widely been documented in circulating cells and plasma of CHF patients, deregulation of epigenetic features could play a key role in disease progression. Here, we review current knowledge of the contribution of EPCs and CACs to endothelial repair mechanisms in CHF patients, focusing on the effects induced by exercise training and hypothesizing that some of these effects can be mediated by epigenetic mechanisms.

Exosome-based immunomodulation during aging: A nano-perspective on inflamm-aging

Exosomes are nanovesicles formed by inward budding of endosomal membranes. They exert complex immunomodulatory effects on target cells, acting both as antigen-presenting vesicles and as shuttles for packets of information such as proteins, coding and non-coding RNA, and nuclear and mitochondrial DNA fragments. Albeit different, all such functions seem to be encompassed in the adaptive mechanism mediating the complex interactions of the organism with a variety of stressors, providing both for defense and for the evolution of symbiotic relationships with others organisms (gut microbiota, bacteria, and viruses). Intriguingly, the newly deciphered human virome and exosome biogenesis seem to share some physical-chemical characteristics and molecular mechanisms. Exosomes are involved in immune system recognition of self from non-self throughout life: they are therefore ideal candidate to modulate inflamm-aging, the chronic, systemic, age-related pro-inflammatory status, which influence the development/progression of the most common age-related diseases (ARDs). Not surprisingly, recent evidence has documented exosomal alteration during aging and in association with ARDs, even though data in this field are still limited. Here, we review current knowledge on exosome-based trafficking between immune cells and self/non-self cells (i.e. the virome), sketching a nano-perspective on inflamm-aging and on the mechanisms involved in health maintenance throughout life.

Immunocytochemical localization of calretinin-containing neurons in the rat periaqueductal gray and colocalization with enzymes producing nitric oxide: a double, double-labeling study.

The pattern of distribution and colocalization of the calcium‐binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH‐diaphorase (NADPH‐d) histochemistry and with NOS immunocytochemistry, respectively. Cal‐immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG‐dl) and ventrolateral PAG (PAG‐vl). Double‐labeled neurons were found only in PAG‐dl. The first experimental approach indicated that 33–41% of the NADPH‐d‐positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH‐d activity appeared in 19–26% of the Cal‐immunopositive (CalIP) neurons. Two‐color immunofluorescence revealed that ∼39–43% of NOS‐immunoreactive (NOSIR) neurons were double‐labeled with Cal and ∼23% of CalIP neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG‐dl, showed that CalIP neurons had a cross‐sectional area of 94.7 μm2, whereas Nadph+ neurons and double‐labeled neurons were slightly smaller, having a cross‐sectional area of 90.5 and 91.4 μm2, respectively. On electron microscopy, CalIP axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially CalIP dendrites. These experiments suggest that PAG‐dl is characterized by a high degree of heterogeneity. Synapse, 2012.

Intracallosal neuronal nitric oxide synthase neurons colocalize with neurokinin 1 substance P receptor in the rat

The corpus callosum (cc) contains nitric oxide (NO)‐producing neurons. Because NO is a potent vasodilator, these neurons could translate neuronal signals into vascular responses that can be detected by functional brain imaging. Substance P (SP), one of the most widely expressed peptides in the CNS, also produces vasomotor responses by inducing calcium release from intracellular stores through its preferred neurokinin 1 (NK1) receptor, thus inducing NO production via activation of neuronal NO synthase (nNOS). Single‐ and double‐labeling experiments were performed to establish whether NK1‐immunopositive neurons (NK1IP‐n) are found in the rat cc and the extent of NK1 colocalization with nNOS. NK1IP‐n were seen to constitute a large neuronal population in the cc and had a distribution similar to that of nNOSIP neurons (nNOSIP‐n). NK1IP‐n were numerous in the lateral cc and gradually decreased in the more medial portions, where they were few or absent. Intracallosal NK1IP‐n and their dendritic trees were intensely labeled, allowing classification into four morphological types: bipolar, round, polygonal, and pyramidal. Confocal microscopic examination demonstrated that nearly all NK1IP‐n contained nNOS (96.43%) and that 84.59% of nNOSIP‐n co‐expressed NK1. These data suggest that the majority of intracallosal neurons can release NO as a result of the action of SP. A small proportion of nNOSIP‐n does not contain NK1 and is not activated by SP; these neurons may release NO via alternative mechanisms. The possible mechanisms by which intracallosal neurons release NO are also reviewed. J. Comp. Neurol. 523:589–607, 2015.

Differential distribution of parvalbumin- and calbindin-D28K-immunoreactive neurons in the rat periaqueductal gray matter and their colocalization with enzymes producing nitric oxide.

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (ParvIP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. CalbIP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 μm(2) (PAG-dl) to 125.13 μm(2) (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence). Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons. Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of CalbIP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOSIP cells colocalized with Calb and 24-34% of CalbIP neurons contained nNOS. DL neuron size was 104.44 μm(2); neurons labeled only with NADPH-d or Calb measured 89.793 μm(2) and 113.48 μm(2), respectively. Together with previous findings (Barbaresi et al. [2012]) these data suggest that: Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved.

Substance P NK1 receptor in the rat corpus callosum during postnatal development

The expression of substance P (SP) receptor (neurokinin 1, NK1) was studied in the rat corpus callosum (cc) from postnatal day 0 (the first 24 hr from birth, P0) to P30.