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Aims Sodium-glucose cotransporter 2 inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and non-diabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. Methods and results Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day), had fewer apoptotic cells (4.9 ± 2.1 vs. 1 ± 0.5 TUNEL-positive cells %, P < 0.05), less senescence (10.1 ± 2 vs. 7.9 ± 1.2 β-gal positivity/tissue area, P < 0.05), fibrosis (0.2 ± 0.05 vs. 0.15 ± 0.06, P < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs. 2.3 ± 0.6 fluorescence intensity/total area, P < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a down-regulation of the 5′ adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to the modulation of cardiomyogenic transcription factors, we analysed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice [0.5 ± 0.01 densitometric units (DU)], non-diabetic mice treated with EMPA (2.2 ± 0.01 DU, P < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, P < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs. 2.2 ± 0.01 target protein/β-actin ratio, P < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer glycogen synthase kinase 3 beta (GSK3β), leading to reactivation of cardiomyogenic transcription factors. Conclusion Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3β signalling pathway in the context of diabetes. [ABSTRACT FROM AUTHOR]

Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren's Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions. [ABSTRACT FROM AUTHOR]

Empagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. To assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. Male C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20 %, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. EMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity. [Display omitted] • Empagliflozin prevented ponatinib-induced cardiotoxicity in vivo and in vitro. • Cx43-autophagy pathway is involved in the empagliflozin's cardiac protective effect. • Empagliflozin restored the SRF-SRE binding and cardiac actin expression. • Gliflozin's effects on cardiotoxicity are worthy of clinical studies. [ABSTRACT FROM AUTHOR]

Gap junctions (GJ) mediate electrical coupling between cardiac myocytes, allowing the spreading of the electrical wave responsible for synchronized contraction. GJ function can be regulated by modulation of connexon densities on membranes, connexin (Cx) phosphorylation, trafficking and degradation. Recent studies have shown that adenosine (A) involves Cx43 turnover in A 1 receptor-dependent manner, and dipyridamole increases GJ coupling and amount of Cx43 in endothelial cells. As the abnormalities in GJ organization and regulation have been described in diseased myocardium, the aim of the present study was to assess the regional expression of molecules involved in GJ regulation in a model of left ventricular dysfunction (LVD). For this purpose the distribution and quantitative expression of Cx43, its phosphorylated form pS368-Cx43, PKC phosphorylated substrates, RhoA and A receptors, were investigated in experimental models of right ventricular-pacing induced LVD, undergoing concomitant dipyridamole therapy or placebo, and compared with those obtained in the myocardium from sham-operated minipigs. Results demonstrate that an altered pattern of factors involved in Cx43-made GJ regulation is present in myocardium of a dysfunctioning left ventricle. Furthermore, dipyridamole treatment, which shows a mild protective role on left ventricular function, seems to act through modulating the expression and activation of these factors as confirmed by in vitro experiments on cardiomyoblastic cell line H9c2 cells. [ABSTRACT FROM AUTHOR]

Introduction: Connexins (Cxs) are transmembrane proteins involved in the formation of connexons/hemichannels. Two connexons of adjacent cells can dock one each other form- ing an intercellular channel at gap junction (GJ), by which ions and small metabolites can pass between cells. Single hemichannels participate in the exchange of small metabolites between cells and the extracellular space [1]. There are at least 21 isoforms of Cxs expressed in humans and they are localized in almost every tissue. More than one Cx can be expressed in each tissue and connexons can be made by a single Cx or by different types of Cxs. GJ are involved in various physiological functions including secretion in glandular tissue [2-5]. Cx26, Cx32 and Cx43 are the mainly expressed Cxs in glands, but no data are available about their expression in human salivary glands. Aim: The aim of our study is to investigate the presence and the localization of these Cxs in human minor salivary glands. Methods: human minor salivary gland biopsies (hMSGBs) were obtained as part of the routine diagnostic procedures when primary Sjögren’s Syndrome (pSS) was suspected. Samples without pSS were selected for the study. Immunofluorescence and immunoelectron microscopy were employed to evaluated Cx26, Cx32 and Cx43 protein expression while RT-PCR was used to detect their mRNA expression. Results and Conclusions: Cx expression was found at both protein and mRNA level in all hMSGBs analysed. Cxs were observed at level of the duct and acinar cell membranes, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting a colocalization of these Cxs in the same connexons. Further studies are necessary to confirm this hypothesis. However, these preliminary results are important because demonstrate for the first time the presence of Cxs in human salivary glands. Moreover, in the few analysed pSS samples we observed an alteration of Cx expression indicating that these proteins may be involved in salivary gland dysfunctions. [ABSTRACT FROM AUTHOR]

Abstract: Tissue engineering scaffolds should be able to reproduce optimal microenvironments in order to support cell attachment, three-dimensional growth, migration and, regarding fibroblasts, must also promote extracellular matrix production. Various bioactive molecules are employed in the preparation of spongy scaffolds to obtain biomimetic matrices by either surface-coating or introducing them into the bulk composition of the biomaterial. The biomimetic properties of a spongy matrix composed of PVA combined with the natural component gelatine were evaluated by culturing human gingival fibroblasts on the scaffold. Cell adhesion, morphology and distribution within the scaffold were assessed by histology and electron microscopy; viability and metabolic activity as well as extracellular matrix production were analyzed by MTT assay, cytochemistry and immunocytochemistry. Fibroblasts interacted positively with PVA/gelatine. They adhered to the PVA/gelatine matrix in which they had good spreading activity and active metabolism; fibroblasts were also able to produce extracellular matrix molecules (type I collagen, fibronectin and laminin) compared to bi-dimensionally grown cells. The in situ creation of a biological matrix by human fibroblasts together with the ability to produce growth factor TGF-β1 and the intracellular signal transduction molecule RhoA, suggests that this kind of PVA/gelatine sponge may represent a suitable support for in vitro extracellular matrix production and connective tissue regeneration. [Copyright &y& Elsevier]

Anti-DNA antibodies and anti-endothelial cell antibodies (AECA) are often detected in systemic lupus erythematosus (SLE). Anti-DNA antibodies can also bind the membrane of human umbilical vein endothelial cells (HUVEC), but little is known about the presence of AECA in the population of immunoglobulins from SLE sera that do not bind DNA. The aim of this study is to analyse the ability of anti-DNA and non-anti-DNA antibodies from SLE sera to bind endothelial cell antigens and to investigate their pathogenic potential. Both anti-DNA and non-anti-DNA antibodies display AECA activity by immunoprecipitation and flow cytometry and in some patients recognize antigens of identical molecular weight. Complement-dependent cytotoxicity on HUVEC was not detected with either anti-DNA or non-anti-DNA antibodies. Similarly, apoptosis was not induced in HUVEC and HL60 incubated with anti-DNA or non-anti-DNA antibodies, as shown by the DNA hypodiploid content. These data indicate that AECA are highly heterogeneous, as they recognize a wide variety of surface molecules on HUVEC and equally present in anti-DNA and non-anti-DNA antibodies from SLE patients. [Copyright &y& Elsevier]

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner. [ABSTRACT FROM AUTHOR]

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity. [ABSTRACT FROM AUTHOR]

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood–brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property. [ABSTRACT FROM AUTHOR]

The article discusses a study conducted to explore the signaling pathway of Pontine (PON) in a murine model of PON-induced cardiotoxicity focusing on the involvement of Cx43, serine 368 phoshorylated Cx43 (pS368-Cx43) and Cx26 also considering sex-related differences. The study found that c onnexins are involved in mice PON-induced cardiotoxicity, showing a sex related altered expression.

• Cx43 and Cx26 proteins are reduced during aging in cardiomyocytes of adult rat. • Cx26 and Cx32 proteins are reduced during aging in hepatocytes of adult rat. • Cx43/Cx26/Cx32 decrease with aging on the cardiomyocytes and hepatocytes membrane. • mRNA of Cx43/Cx26/Cx32 does not change during early aging in heart and liver. Connexins are membrane-spanning proteins that form membrane channels and hemichannels. They are involved in the cellular communication and in the maintenance of tissue homeostasis. Recent studies in humans and animals have demonstrated that the expression and distribution of Cx43, the most studied connexin, can change during aging. However, the research on the involvement of the other connexins in cardiac and hepatic aging is, at present, still very poor. Hence, the aim of this study is to evaluate the expression of Cx43 and Cx26 in the heart as well as Cx26 and Cx32 in the liver of a rat model that aged naturally, rather than prematurely because of genetic mutations or age-related diseases. The results obtained in the present study have demonstrated that these connexins decrease in rat cardiomyocytes and in rat hepatocytes as they age. This change was revealed only at protein level, as connexin-mRNAs remained unchanged during aging. Moreover, the aged rats showed an increase in body fat, whose subcutaneous layer tended to be higher. Finally, how these changes could represent signs of physiological adaptation in successful aging was discussed. [ABSTRACT FROM AUTHOR]

Mood alterations, anxiety, and cognitive impairments associated with adult-onset hypothyroidism often persist despite replacement treatment. In rodent models of hypothyroidism, replacement does not bring 3-iodothyronamine (T1AM) brain levels back to normal. T1AM is a thyroid hormone derivative with cognitive effects. Using a pharmacological hypothyroid mouse model, we investigated whether augmenting levothyroxine (L-T4) with T1AM improves behavioural correlates of depression, anxiety, and memory and has an effect on hippocampal neurogenesis. Hypothyroid mice showed impaired performance in the novel object recognition test as compared to euthyroid mice (discrimination index (DI): 0.02 ± 0.09 vs. 0.29 ± 0.06; t = 2.515, p = 0.02). L-T4 and L-T4+T1AM rescued memory (DI: 0.27 ± 0.08 and 0.34 ± 0.08, respectively), while T1AM had no effect (DI: −0.01 ± 0.10). Hypothyroidism reduced the number of neuroprogenitors in hippocampal neurogenic niches by 20%. L-T4 rescued the number of neuroprogenitors (mean diff = 106.9 ± 21.40, t = 4.99, pcorr = 0.003), while L-T4+T1AM produced a 30.61% rebound relative to euthyroid state (mean diff = 141.6 ± 31.91, t = 4.44, pcorr = 0.004). We performed qPCR analysis of 88 genes involved in neurotrophic signalling pathways and found an effect of treatment on the expression of Ngf, Kdr, Kit, L1cam, Ntf3, Mapk3, and Neurog2. Our data confirm that L-T4 is necessary and sufficient for recovering memory and hippocampal neurogenesis deficits associated with hypothyroidism, while we found no evidence to support the role of non-canonical TH signalling. [ABSTRACT FROM AUTHOR]

The aim of the present research was to explore the mechanisms underlying the role of dopamine in the regulation of insulin secretion in beta cells. The effect of dopamine on insulin secretion was investigated on INS 832/13 cell line upon glucose and other secretagogues stimulation. Results show that dopamine significantly inhibits insulin secretion stimulated by both glucose and other secretagogues, while it has no effect on the basal secretion. This effect requires the presence of dopamine during incubation with the various secretagogues. Both electron microscopy and immunohistochemistry indicate that in beta cells the D 2 dopamine receptor is localized within the insulin granules. Blocking dopamine entry into the insulin granules by inhibiting the VMAT2 transporter with tetrabenazine causes a significant increase in ROS production. Our results confirm that dopamine plays an important role in the regulation of insulin secretion by pancreatic beta cells through a regulated and precise compartmentalization mechanisms. [Display omitted] • Pancreatic beta cells and dopaminergic neurons share several biochemical and functional characteristics. • Dopamine significantly inhibits insulin secretion stimulated by both glucose and other secretagogues (KCl and IBMX). • This effect is closely regulated by a precise compartmentalization mechanism. • The inhibition of this mechanism increases ROS production and counteract the negative effect of dopamine on GSIS. [ABSTRACT FROM AUTHOR]

eNOS‐deficient mice were previously shown to develop hypertension and metabolic alterations associated with insulin resistance either in standard dietary conditions (eNOS−/− homozygotes) or upon high‐fat diet (HFD) (eNOS+/− heterozygotes). In the latter heterozygote model, the present study investigated the pancreatic morphological changes underlying the abnormal glycometabolic phenotype. C57BL6 wild type (WT) and eNOS+/− mice were fed with either chow or HFD for 16 weeks. After being longitudinally monitored for their metabolic state after 8 and 16 weeks of diet, mice were euthanized and fragments of pancreas were processed for histological, immuno‐histochemical and ultrastructural analyses. HFD‐fed WT and eNOS+/− mice developed progressive glucose intolerance and insulin resistance. Differently from WT animals, eNOS+/− mice showed a blunted insulin response to a glucose load, regardless of the diet regimen. Such dysregulation of insulin secretion was associated with pancreatic β‐cell hyperplasia, as shown by larger islet fractional area and β‐cell mass, and higher number of extra‐islet β‐cell clusters than in chow‐fed WT animals. In addition, only in the pancreas of HFD‐fed eNOS+/− mice, there was ultrastructural evidence of a number of hybrid acinar‐β‐cells, simultaneously containing zymogen and insulin granules, suggesting the occurrence of a direct exocrine‐endocrine transdifferentiation process, plausibly triggered by metabolic stress associated to deficient endothelial NO production. As suggested by confocal immunofluorescence analysis of pancreatic histological sections, inhibition of Notch‐1 signaling, likely due to a reduced NO availability, is proposed as a novel mechanism that could favor both β‐cell hyperplasia and acinar‐β‐cell transdifferentiation in eNOS‐deficient mice with impaired insulin response to a glucose load. [ABSTRACT FROM AUTHOR]

We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly (ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs. [ABSTRACT FROM AUTHOR]

In this paper we investigated a novel and non-invasive approach for an endogenous osteoblast stimulation mediated by boron nitride nanotubes (BNNTs). Specifically, following the cellular uptake of the piezoelectric nanotubes, cultures of primary human osteoblasts (hOBs) were irradiated with low frequency ultrasound (US), as a simple method to apply a mechanical input to the cells loaded with BNNTs. This in vitro study was aimed at investigating the main interactions between hOBs and BNNTs and to study the effects of the ‘BNNTs + US’ stimulatory method on the osteoblastic function and maturation.A non-cytotoxic BNNT concentration to be used in vitro with hOB cultures was established. Moreover, investigation with transmission electron microscopy/electron energy loss spectroscopy (TEM/EELS) confirmed that BNNTs were internalized in membranal vesicles. The panel of investigated osteoblastic markers disclosed that BNNTs were capable of fostering the expression of late-stage bone proteins in vitro, without using any mineralizing culture supplements. In our samples, the maximal osteopontin expression, with the highest osteocalcin and Ca2+ production, in the presence of mineral matrix with nodular morphology, was observed in the samples treated with BNNTs + US. In this group was also shown a significantly enhanced synthesis of TGF-β1, a molecule sensitive to electric stimulation in bone. Finally, gene deregulations of the analyzed osteoblastic genes leading to depletive cellular effects were not detected. Due to their piezoelectricity, BNNT-based therapies might disclose advancements in the treatment of bone diseases. [ABSTRACT FROM AUTHOR]

Ponatinib (PON), a third-generation tyrosine kinase inhibitor (TKI), has proven cardiovascular toxicity, with no known preventing agents usable to limit such side effect. Sodium-glucose cotransporter type 2 (SGLT2) inhibitors are a new class of glucose-lowering agents, featuring favorable cardiac and vascular effects. We assessed the effects of the SGLT2 inhibitors empagliflozin (EMPA) and dapagliflozin (DAPA) on human aortic endothelial cells (HAECs) and underlying vasculo-protective mechanisms in an in vitro model of PON-induced endothelial toxicity. We exposed HAECs to PON or vehicle (DMSO) in the presence or absence of EMPA (100 and 500 nmol/L) or dapagliflozin (DAPA) for 0–48 h exposure times. Compared with vehicle, incubations of HAECs with PON significantly reduced cell viability (0.56 ± 0.11 vs 0.23 ± 0.05 absorbance units, p < 0.01), increased the number of senescent cells at β-gal-assay (PON 9 ± 4 vs basal DMSO 3 ± 1 β-Gal+ cells/field, p < 0.01), decreased tubulization in Matrigel (PON PON: 6 ± 1 vs basal DMSO 12 ± 1 tubuli number/field, p < 0.05) with a non-statistically significant trend of PON to decrease the number of autophagic cells at immunofluorescence assay and flow cytometry. EMPA reverted the effects of PON on cell viability (E 500 + PON 0.24 ± 0.05 vs PON 0.56 ± 0.11 absorbance units, p < 0.01) and induced autophagy (E 500 7 ± 4.3 vs basal DMSO 2.6 ± 2.3 mean fluorescence vs PON 2.6 ± 2.4 mean fluorescence, p < 0.05). EMPA and DAPA also reversed the effects of PON on cell senescence (E 500 + PON 4 ± 1 and DAPA 100 4 ± 2 vs PON 9 ± 4 β-Gal+ cells/field, p < 0.01) and improved cell tubulization (E 500 + PON 21 ± 3 vs PON 6 ± 1 tubuli number/field, p < 0.05; DAPA 100 + PON 16 ± 2 vs PON 6 ± 1 tubuli number/field, p < 0.05). EMPA and DAPA attenuate the vasculo-toxic effect exerted by PON by reverting endothelial cell senescence and dysfunction. These findings support the design of clinical studies exploring the vasculo-protective effects of EMPA or DAPA on PON-induced vascular toxicity. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this research was to characterize the immediate alterations induced by angioplasty and to compare the results of the application of two types of balloons. Ten porcine renal arteries were dilated with a compliant balloon, and ten with a non-compliant balloon. After angioplastic treatment arterial specimens were wax embedded for light microscopy. Sections were stained with the orcein-Van Gieson method, orcein, haematoxylin–eosin, and PAS. Image analysis was performed taking into consideration the following parameters: thickness of the entire wall, of the tunica media and of the inner elastic lamina. The major axes of the smooth muscle cells nuclei were also measured. The effects of the two types of balloon resulted in changes consisting in thinning of the entire arterial wall, reduction of the tunica media, distension of reticular fibers, presence of wide spaces between smooth muscle cells, stretching of smooth muscle cells, inner elastic lamina thickening. Both angioplasty devices used can modify the vascular wall. The identification of the tunica media structural damages might be useful in order to estimate the behavior of the vascular wall in the follow-up after angioplasty, because the entity of modifications could be predictive of restenosis that often takes place weeks or months after angioplasty. [Copyright &y& Elsevier]

Introduction: Naturally occurring antibodies (auto-Abs) recognizing human granulocyte-colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. Materials and methods: Mononuclear cells from cord blood samples of di3erent newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. Results: Six di3erent, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD5-, CD10-, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing λ clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. Conclusion: The secreted Abs did not a3ect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing. [ABSTRACT FROM AUTHOR]

Background Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System. Methods Bone marrow aspirates were obtained from healthy adult individuals undergoing routine total hip replacement surgery and used to generate primary cultures in the bioreactor. HPL and ABS were tested as supplements to culture medium. Morphological observations, cytofluorimetric analysis, lactate and glucose level assessment were performed. Results ABS gave rise to both heterogeneous MSC and MPC population. About 95% of cells cultured in HPL showed a fibroblast-like morphology and typical mesenchymal surface markers, but MPCs were scarcely represented. Discussion The use of ABS appeared to sustain a large scale MSC production, as well as the recovery of a subset of MPCs, and resulted a suitable alternative to HPL in the cell generation based on the Quantum System. [ABSTRACT FROM AUTHOR]

The ideal scaffold for bone regeneration is required to be highly porous, non-immunogenic, biostable until the new tissue formation, bioresorbable and osteoconductive. This study aimed at investigating the process of new bone formation in patients treated with granular SmartBone ® for sinus augmentation, providing an extensive histologic analysis. Five biopsies were collected at 4–9 months post SmartBone ® implantation and processed for histochemistry and immunohistochemistry. Histomorphometric analysis was performed. Bone-particle conductivity index (BPCi) was used to assess SmartBone ® osteoconductivity. At 4 months, SmartBone ® (12%) and new bone (43.9%) were both present and surrounded by vascularized connective tissue (37.2%). New bone was grown on SmartBone ® (BPCi = 0.22). At 6 months, SmartBone ® was almost completely resorbed (0.5%) and new bone was massively present (80.8%). At 7 and 9 months, new bone accounted for a large volume fraction (79.3% and 67.4%, respectively) and SmartBone ® was resorbed (0.5% and 0%, respectively). Well-oriented lamellae and bone scars, typical of mature bone, were observed. In all the biopsies, bone matrix biomolecules and active osteoblasts were visible. The absence of inflammatory cells confirmed SmartBone ® biocompatibility and non-immunogenicity. These data indicate that SmartBone ® is osteoconductive, promotes fast bone regeneration, leading to mature bone formation in about 7 months. [ABSTRACT FROM AUTHOR]

In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine. [ABSTRACT FROM AUTHOR]

Abstract: Thanks to a non-covalent wrapping with glycol-chitosan, highly biocompatible and highly concentrated dispersions of boron nitride nanotubes were obtained and tested on human neuroblastoma cells. A systematic investigation of the cytotoxicity of these nanovectors with several complementary qualitative and quantitative assays allowed a strong interference with the MTT metabolic assay to be highlighted, similar to a phenomenon already observed for carbon nanotubes, that would wrongly suggest toxicity of boron nitride nanotubes. These results confirm the high complexity of these new nanomaterials, and the needing of extensive investigations on their exciting potential applications in the biomedical field. [Copyright &y& Elsevier]

The analysis of myenteric neurons is becoming increasingly important for the assessment of enteric nervous system injury and degeneration occurring in motor disorders of the gut. Limited information is presently available on the quantitative estimation of myenteric neurons and glial cells in paraffin-embedded colonic sections; additional data would be useful for diagnostic purposes. In this morphometric study, we performed immunohistochemistry to count myenteric neurons and glial cells in paraffin sections of human colon. Serial cross sections of formalin-fixed paraffin-embedded full-thickness normal human left colon ( n = 10, age-range: 50–72 years) were examined. HuC/D and S100β antigens were found to be the best markers for the detection of neurons and glial cells, respectively. Significant correlations were noted between the numbers of neurons/glial cells and the respective myenteric ganglion areas. These findings suggest that HuC/D-S100β-immunostained paraffin cross sections of human colon can be regarded as valuable tools for the quantitative estimation of myenteric neurons and glial cells. Based on the present method, only a limited number of paraffin sections are needed for reliable quantitative assessments of myenteric ganglion cells, thus allowing fast and simple approaches in the settings of the histopathological diagnosis of colonic motility disorders and retrospective evaluations of pathological archival tissue specimens. [ABSTRACT FROM AUTHOR]

Background: The Rho/transforming growth factor-β (TGF-β) system plays a crucial role in the progression of renal damage due to stimulation of extracellular matrix molecule deposition. In fact, the in vitro TGF-β-mediated production of fibronectin, one of the major TGF-β-regulated extracellular components, has recently been correlated with Rho protein signalling molecules. Although a close relationship between increased renal tissue levels of TGF-β1 and fibronectin has been reported in IgA nephropathy, no data are available on renal tissue expression of Rho proteins. Methods: This study was designed to assess in IgA nephropathy patients the kidney tissue immunohistochemical expression of RhoA, TGF-β1, and fibronectin, and the rate of immunoreactivity for each antigen by image analysis. Results: An increase in RhoA, TGF-β1, and fibronectin expression was detected in tubulointerstitium and in glomeruli of IgA nephropathy compared to normal kidneys; in particular, RhoA was found also in proximal tubules, unlike control kidneys and mainly at the cell boundary level, which is in keeping with its activated form. The image analysis confirmed that the kidney tissue levels of RhoA, TGF-β1, and fibronectin were significantly enhanced in the patients. Conclusion: This study suggests that RhoA may represent a key molecule in the signalling transduction pathway of profibrotic signals in IgA nephropathy. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Sjögren's syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5–ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5–ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5–ezrin co-localization. Our data reveal that AQP5–ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients [ABSTRACT FROM AUTHOR]

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers. [ABSTRACT FROM AUTHOR]

Peptide oral administration is a hard goal to reach, especially if the brain is the target site. The purpose of the present study was to set up a vehicle apt to promote oral absorption of the neuropeptide dalargin (DAL), allowing it to cross the intestinal mucosal barrier, resist enzymatic degradation, and transport drugs to the brain after crossing the blood–brain barrier. Therefore, a chitosan quaternary ammonium derivative was synthesized and conjugated with methyl-β-cyclodextrin to prepare DAL-medicated nanoparticles (DAL-NP). DAL-NP particle size was 227.7 nm, zeta potential +8.60 mV, encapsulation efficiency 89%. DAL-NP protected DAL from degradation by chymotrypsin or pancreatin and tripled DAL degradation time compared to non-encapsulated DAL. Use of DAL-NP was safe for either Caco-2 or bEnd.3 cells, with the latter selected as a blood–brain barrier model. DAL-NP could also cross either the Caco-2 or bEnd.3 monolayer by the transepithelial route. The results suggest a potential DAL-NP ability to transport to the brain a DAL dose fraction administered orally, although in vivo experiments will be needed to confirm the present data obtained in vitro. [ABSTRACT FROM AUTHOR]

Osteopontin (OPN), a novel hepatic damage marker, as well as several non-coding RNA seem to be associated with liver aging, a little studied and not yet defined process. The aim of the study was to evaluate the expression variations of OPN and miRNA-181a , the transcriptional profiling of long non coding (lnc) RNA GAS-5 / miRNA-222 axis and lncRNA NEAT-1 in liver tissue of rats. In addition, to monitor the senescence process, the telomere shortening and TERT/TERC gene expression were also measured. Three groups of male Wistar rats were studied: A (n = 6, young); B (n = 13, adult); C (n = 10, old). Total RNA, including miRNAs, was extracted from liver and analysed by Real-Time PCR. Ultrasound and biochemical evaluation were performed in all rats as well as the histological analysis. OPN mRNA resulted lower in C with respect to A and B while miRNA-181a expression was significantly increased as a function of age. An increasing of both NEAT -1 and miRNA-222 expression as a function of age in parallel with a decreasing of GAS-5 expression in young and old rats, but not in the adults, was observed. A positive correlation was detected between miRNA-181a and miRNA-222. The hepatic ultrasound analysis revealed areas of hyperechogenicity distributed as a function of age. A significant telomere shortening was measured as a function of age while the two subunits TERT and TERC expressions showed an opposite trend. This work could provide a valid starting point to better understand the physiopathological changes during aging, pinpointing in the OPN / miRNA-181a axis significant predictors of successful aging. Unlabelled Image • Aging is attended by a low-grade systemic inflammation. • OPN counter-regulation mediated by miR-181a : hepatic markers of inflamm-aging • GAS-5 is a target of miRNA-222 : reciprocal repression regulatory loop. • Low-grade inflammation associated with development of age-related conditions • Multivariate analysis: OPN/miRNA-181a significant predictors of successful aging [ABSTRACT FROM AUTHOR]

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage. [ABSTRACT FROM AUTHOR]