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Melanoma is a relative rare disease worldwide, nevertheless it has a great relevance in some countries such as in Europe. In order to shed some light upon the transcriptional profile of skin melanoma, we compared the gene expression of 6 independent tumours (all progressed towards metastatic disease and with wild type BRAF) to the expression profile of healthy melanocytes. Paraffin-embedded samples were manually microdissected to obtain enriched samples and then RNA was extracted and analysed through a microarray-based approach. An exhaustive bioinformatics analysis was performed to identify differentially expressed transcripts between the two groups as well as enriched functional terms. Overall, 50 up- and 19 down-regulated transcripts were found to be significantly changed in tumour compared to control tissue. Among the upregulated transcripts, the majority belonged to the immune response group and to proteasome, while most of the downregulated genes were related to cytosolic ribosomes. Interestingly, Gene Set Enrichment Analysis (GSEA), along with the analysis of the expression data retrieved from TCGA/GTEx databases, confirmed the general trend of downregulation affecting cytoribosome proteins. In contrast, transcripts coding for mitori-bosome proteins showed the opposite trend.

Because of its high incidence and poor prognosis, colorectal cancer (CRC) represents an important health issue in several countries. As with other carcinomas, the so-called tumour microenvironment (TME) has been shown to play key roles in CRC progression and related therapeutical outcomes, even though a deeper understanding of the underlying molecular mechanisms is needed to devise new treatment strategies. For some years now, omics technologies and consolidated bioinformatics pipelines have allowed scientists to access large amounts of biologically relevant information, even when starting from small tissue samples, thus, in order to shed new light upon the role of the TME in CRC, we compared the gene expression profiles of 6 independent tumour tissues (all progressed towards metastatic disease) to the expression profile of the surrounding stromata. To do this, paraffin-embedded whole tissues were first microdissected to obtain samples enriched with tumour and stromal cells, respectively. Afterwards, RNA was extracted and analysed using a microarray-based approach. A thorough bioinformatics analysis was then carried out to identify transcripts differentially expressed between the two groups and possibly enriched functional terms. Overall, 193 genes were found to be significantly downregulated in tumours compared to the paired stromata. The functional analysis of the downregulated gene list revealed three principal macro areas of interest: the extracellular matrix, cell migration, and angiogenesis. Conversely, among the upregulated genes, the main alterations detected by the functional annotation were related to the ribosomal proteins (rProteins) of both the large (60S) and small (40S) subunits of the cytosolic ribosomes. Subsequent gene set enrichment analysis (GSEA) confirmed the massive overexpression of most cytosolic—but not mitochondrial—ribosome rProteins.

Proanthocyanidins (PACs) are a class of polyphenolic compounds that are attracting considerable interest in the nutraceutical field due to their potential health benefits. However, knowledge about the chemistry, biosynthesis, and distribution of PACs is limited. This review summarizes the main chemical characteristics and biosynthetic pathways and the main analytical methods aimed at their identification and quantification in raw plant matrices. Furthermore, meta-analytic approaches were used to identify the main plant sources in which PACs were contained and to investigate their potential effect on human health. In particular, a cluster analysis identified PACs in 35 different plant families and 60 different plant parts normally consumed in the human diet. On the other hand, a literature search, coupled with forest plot analyses, highlighted how PACs can be actively involved in both local and systemic effects. Finally, the potential mechanisms of action through which PACs may impact human health were investigated, focusing on their systemic hypoglycemic and lipid-lowering effects and their local anti-inflammatory actions on the intestinal epithelium. Overall, this review may be considered a complete report in which chemical, biosynthetic, ecological, and pharmacological aspects of PACs are discussed.

Aim: To clarify the mechanisms of interaction between SiO2 nanoparticles (NPs) and the plasma membrane of GT1–7 neuroendocrine cells, with focus on the activation of calcium-permeable channels, responsible for the long lasting calcium influx and modulation of the electrical activity in these cells. Materials & methods: Nontoxic doses of SiO2 NPs were administered to the cells. Calcium imaging and patch clamp techniques were combined with a pharmacological approach. Results: TRPV4, Cx and Panx-like channels are the major components of the NP-induced inward currents. Preincubation with the antioxidant N-acetyl-L-cysteine strongly reduced the [Ca2+]i increase. Conclusion: These findings suggest that SiO2 NPs directly activate a complex set of calcium-permeable channels, possibly by catalyzing free radical production.

In-depth characterization of heart-brain communication in critically ill patients with severe acute respiratory failure is attracting significant interest in the COronaVIrus Disease 19 (COVID-19) pandemic era during intensive care unit (ICU) stay and after ICU or hospital discharge. Emerging research has provided new insights into pathogenic role of the deregulation of the heart-brain axis (HBA), a bidirectional flow of information, in leading to severe multiorgan disease syndrome (MODS) in patients with confirmed infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Noteworthy, HBA dysfunction may worsen the outcome of the COVID-19 patients. In this review, we discuss the critical role HBA plays in both promoting and limiting MODS in COVID-19. We also highlight the role of HBA as new target for novel therapeutic strategies in COVID-19 in order to open new translational frontiers of care. This is a translational perspective from the Italian Society of Cardiovascular Researches., Graphical Abstract ga1

The crosstalk among cancer cells (CCs) and stromal cells within the tumor microenvironment (TME) has a prominent role in cancer progression. The significance of endothelial cells (ECs) in this scenario relies on multiple vascular functions. By forming new blood vessels, ECs support tumor growth. In addition to their angiogenic properties, tumor-associated ECs (TECs) establish a unique vascular niche that actively modulates cancer development by shuttling a selected pattern of factors and metabolites to the CC. The profile of secreted metabolites is strictly dependent on the metabolic status of the cell, which is markedly perturbed in TECs. Recent evidence highlights the involvement of heme metabolism in the regulation of energy metabolism in TECs. The present study shows that interfering with endothelial heme metabolism by targeting the cell membrane heme exporter Feline Leukemia Virus subgroup C Receptor 1a (FLVCR1a) in TECs, resulted in enhanced fatty acid oxidation (FAO). Moreover, FAO-derived acetyl-CoA was partly consumed through ketogenesis, resulting in ketone bodies (KBs) accumulation in FLVCR1a-deficient TECs. Finally, the results from this study also demonstrate that TECs-derived KBs can be secreted in the extracellular environment, inducing a metabolic rewiring in the CC. Taken together, these data may contribute to finding new metabolic vulnerabilities for cancer therapy.

Hypercholesterolemia is one of the major causes of cardiovascular disease, the risk of which is further increased if other forms of dyslipidemia occur. Current therapeutic strategies include changes in lifestyle coupled with drug administration. Statins represent the most common therapeutic approach, but they may be insufficient due to the onset of resistance mechanisms and side effects. Consequently, patients with mild hypercholesterolemia prefer the use of food supplements since these are perceived to be safer. Here, we investigate the phytochemical profile and cholesterol-lowering potential of Protium heptaphyllum gum resin extract (PHE). Chemical characterization via HPLC-APCI-HRMS2 and GC-FID/MS identified 13 compounds mainly belonging to ursane, oleanane, and tirucallane groups. Studies on human hepatocytes have revealed how PHE is able to reduce cholesterol production and regulate the expression of proteins involved in its metabolism. (HMGCR, PCSK9, LDLR, FXR, IDOL, and PPAR). Moreover, measuring the inhibitory activity of PHE against HMGR, moderate inhibition was recorded. Finally, molecular docking studies identified acidic tetra- and pentacyclic triterpenoids as the main compounds responsible for this action. In conclusion, our study demonstrates how PHE may be a useful alternative to contrast hypercholesterolemia, highlighting its potential as a sustainable multitarget natural extract for the nutraceutical industry that is rapidly gaining acceptance as a source of health-promoting compounds.

The use of mesenchymal stem cells (MSCs) for regenerative purposes has become common in a large variety of diseases. In the dental and maxillofacial field, there are emerging clinical needs that could benefit from MSC-based therapeutic approaches. Even though MSCs can be isolated from different tissues, such as bone marrow, adipose tissue, etc., and are known for their multilineage differentiation, their different anatomical origin can affect the capability to differentiate into a specific tissue. For instance, MSCs isolated from the oral cavity might be more effective than adipose-derived stem cells (ASCs) for the treatment of dental defects. Indeed, in the oral cavity, there are different sources of MSCs that have been individually proposed as promising candidates for tissue engineering protocols. The therapeutic strategy based on MSCs can be direct, by using cells as components of the tissue to be regenerated, or indirect, aimed at delivering local growth factors, cytokines, and chemokines produced by the MSCs. Here, the authors outline the major sources of mesenchymal stem cells attainable from the oral cavity and discuss their possible usage in some of the most compelling therapeutic frontiers, such as periodontal disease and dental pulp regeneration.

The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells following binding with the cell surface ACE2 receptors, thereby leading to coronavirus disease 2019 (COVID-19). SARS-CoV-2 causes viral pneumonia with additional extrapulmonary manifestations and major complications, including acute myocardial injury, arrhythmia, and shock mainly in elderly patients. Furthermore, patients with existing cardiovascular comorbidities, such as hypertension and coronary heart disease, have a worse clinical outcome following contraction of the viral illness. A striking feature of COVID-19 pandemics is the high incidence of fatalities in advanced aged patients: this might be due to the prevalence of frailty and cardiovascular disease increase with age due to endothelial dysfunction and loss of endogenous cardioprotective mechanisms. Although experimental evidence on this topic is still at its infancy, the aim of this position paper is to hypothesize and discuss more suggestive cellular and molecular mechanisms whereby SARS-CoV-2 may lead to detrimental consequences to the cardiovascular system. We will focus on aging, cytokine storm, NLRP3/inflammasome, hypoxemia, and air pollution, which is an emerging cardiovascular risk factor associated with rapid urbanization and globalization. We will finally discuss the impact of clinically available CV drugs on the clinical course of COVID-19 patients. Understanding the role played by SARS-CoV2 on the CV system is indeed mandatory to get further insights into COVID-19 pathogenesis and to design a therapeutic strategy of cardio-protection for frail patients.

Hair loss is a disorder in which the hair falls out from skin areas such as the scalp and the body. Several studies suggest the use of herbal medicine to treat related disorders, including alopecia. Dermal microcirculation is essential for hair maintenance, and an insufficient blood supply can lead to hair follicles (HF) diseases. This work aims to provide an insight into the ethnohistorical records of some nutritional compounds containing flavonoids for their potential beneficial features in repairing or recovering from hair follicle disruption. We started from a query for “alopecia” OR “hair loss” AND “Panax ginseng C.A. Mey.“ (or other six botanicals) terms included in Pubmed and Web of Sciences articles. The activities of seven common botanicals introduced with diet (Panax ginseng C.A. Mey., Malus pumila Mill cultivar Annurca, Coffea arabica, Allium sativum L., Camellia sinensis (L.) Kuntze, Rosmarinum officinalis L., Capsicum annum L.) are discussed, which are believed to reduce the rate of hair loss or stimulate new hair growth. In this review, we pay our attention on the molecular mechanisms underlying the bioactivity of the aforementioned nutritional compounds in vivo, ex vivo and in vitro studies. There is a need for systematic evaluation of the most commonly used plants to confirm their anti-hair loss power, identify possible mechanisms of action, and recommend their best adoption.

Calcium (Ca2+)-permeable channels are key players in different processes leading to blood vessel formation via sprouting angiogenesis, including endothelial cell (EC) proliferation and migration, as well as in controlling vascular features which are typical of the tumor vasculature.In this review we present an up-to-date and critical view on the role of Ca2+-permeable channels in tumor vascularization, emphasizing on the dual communication between growth factors (mainly VEGF) and Ca2+ signals. Due to the complexity of the tumor microenvironment (TME) as a source of multiple stimuli acting on the endothelium, we aim to discuss the close interaction between chemical and physical challenges (hypoxia, oxidative stress, mechanical stress) and endothelial Ca2+-permeable channels, focusing on transient receptor potential (TRP), store-operated Ca2+ channels (SOCs), and mechanosensitive Piezo channels. This approach will depict their crucial contribution in regulating key properties of tumor blood vessels, such as recruitment of endothelial progenitors cells (EPCs) in the early steps of tumor vascularization, abnormal EC migration and proliferation, and increased vascular permeability. Graphical abstract depicting the functional role of Ca2+-permeable TRP, SOCs and Piezo channels in the biological processes regulating tumor angiogenesis in presence of both chemical (oxidative stress and oxygen levels) and mechanical stimuli (ECM stiffness). SOCs store-operated Ca2+ channels, TRPA transient receptor potential ankyrin, TRPV transient receptor potential vanilloid, TRPC transient receptor potential canonical, TRPM transient receptor potential melastatin, TRPM transient receptor potential vanilloid, O2 oxygen, ECM extracellular matrix.

Current treatments for hair follicle (HF) disruption are based on 5-α reductase inhibitors and prostaglandin modulators. Botanicals and nutraceutical compounds interfere with hair loss or stimulate its partial regrowth. Here, we used in vitro cocultures to investigate the activity of Serenoa repens ( SR) and N-acetyl glucosamine + milk proteins (NAG/Lac) on the paracrine interactions between human microvascular endothelial cells (HMVEC) and HF dermal papilla cells (FDPC). Both SR and NAG/Lac-induced endothelial tubulogenesis were enhanced by FDPC. SR promoted proliferation of both the cell types, while NAG/Lac was effective on endothelium. Vascular endothelial growth factor production, enhanced by SR, was further augmented by FDPC. In FDPC 5-α reductase-II and β-catenin expressions were modified by SR and less by NAG/Lac, with no additional effect by HMVEC. SR and NAG/Lac prevented lipid peroxidation, whereas NAG/Lac was effective on interleukin 1β production. Finally, SR and NAG/Lac differentially affected HMVEC permeability and tight junction proteins content. These data provide a mechanistic background for the potential use of these compounds as promoters of HF vascularization.

Background Massive vessel recruitment is required to sustain rapid tumor growth by delivering oxygen and nutrients. Current strategies to counteract angiogenesis are mostly aimed at reducing tumor vessel density. However, many of these drugs have been shown to trigger hypoxia, thus exacerbating tumor aggressiveness. Promising results come from a completely different approach based on the "normalization" of the endothelial layer and the consequent improvement of the vascular function. This new strategy would ameliorate drug delivery to the tumor meanwhile reducing invasiveness and metastatisation. Objective Since endothelial metabolism has proved essential in the regulation of the angiogenic switch, many recent patents focus on agents able to inhibit specific metabolic pathways in Tumor- Associated Endothelial Cells (TECs) in order to provide vessel normalization. Here, we provide a review of the recent advances in the development of patents on agents targeting endothelial metabolism that have proved effective in several vascular disorders. Methods Results of genetic and pharmacologic studies that brought to the development of patents for methods to counteract aberrant angiogenesis were analysed and sub-divided according to the specific metabolic pathway targeted. Results Growing evidences indicate that targeting specific molecular players involved in the endothelial metabolic remodelling required to sustain aberrant angiogenesis, is a valuable therapeutic strategy that can be exploited in vascular disorders as well as in tumor angiogenesis. Conclusion These findings might have important implications in clinics and could be particularly relevant to patients developing resistance to traditional anti-angiogenic drugs.

Purpose This research aimed to assess whether pink-shaded anodized surfaces could enhance the adhesion of soft tissue cells compared with untreated machined titanium surfaces. Materials and methods Two types of Ti-Al-V titanium samples were prepared: machined titanium (Ti) and anodized titanium (AnoTi). The microstructure was studied by means of a scanning electron microscope. X-ray photoelectron spectroscopy (XPS) was carried out as well. The wetting properties were investigated by the sessile drop technique with water and diiodomethane. To investigate the biologic response in vitro, the epithelial cell line HaCaT and the fibroblastic cell line NHDF were used. Cell adhesion, morphology, and proliferation were evaluated. Results The microstructure of the tested surfaces was irregularly smooth for both types of samples with no relevant morphologic differences. The XPS and HR-XPS performed on the AnoTi samples confirmed the presence of Ti, O, and C, along with Ti oxides. Following the optical contact angle measurements, the anodization process induced a slight transition toward the hydrophobic regime. Consequently, the surface free energy values differed significantly between the anodized and the machined samples. Anodized Ti significantly increased the adhesion and proliferation of both epithelial cells and fibroblasts when compared with the pristine Ti controls. Conclusion Compared with the clinical standard, anodized surfaces could enhance the adhesion of the two major cell types within the peri-implant soft tissues, which makes pink anodization a promising option for implant dentistry.

Silicon-based materials present a high potential for dental implant applications, since silicon has been proven necessary for the correct bone formation in animals and humans. Notably, the addition of silicon is effective to enhance the bioactivity of hydroxyapatite and other biomaterials. The present work aims to expand the knowledge of the role exerted by hydrogen in the biological interaction of silicon-based materials, comparing two hydrogenated amorphous silicon coatings, with different hydrogen content, as means to enhance soft tissue cell adhesion. To accomplish this task, the films were produced by plasma enhanced chemical vapor deposition (PECVD) on titanium substrates and their surface composition and hydrogen content were analyzed by means of X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectrophotometry (FTIR) respectively. The surface energy and roughness were measured through optical contact angle analysis (OCA) and high-resolution mechanical profilometry respectively. Coated surfaces showed a slightly lower roughness, compared to bare titanium samples, regardless of the hydrogen content. The early cell responses of human keratinocytes and fibroblasts were tested on the above mentioned surface modifications, in terms of cell adhesion, viability and morphometrical assessment. Films with lower hydrogen content were endowed with a surface energy comparable to the titanium surfaces. Films with higher hydrogen incorporation displayed a lower surface oxidation and a considerably lower surface energy, compared to the less hydrogenated samples. As regards mean cell area and focal adhesion density, both a-Si coatings influenced fibroblasts, but had no significant effects on keratinocytes. On the contrary, hydrogen-rich films increased manifolds the adhesion and viability of keratinocytes, but not of fibroblasts, suggesting a selective biological effect on these cells.

Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial-specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis and provide evidence that endoplasmic reticulum (ER) stress precedes heme-induced paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. Interfering with endothelial heme export represents a valuable model for a deeper understanding of the molecular mechanisms underlying heme-triggered paraptosis and, in the future, might provide a novel tool for the modulation of angiogenesis in pathophysiologic conditions.

Endothelial cells present in tumors show different origin, phenotype, and genotype with respect to the normal counterpart. Various mechanisms of intra-tumor vasculogenesis sustain the complexity of tumor vasculature, which can be further modified by signals deriving from the tumor microenvironment. As a result, resistance to anti-VEGF therapy and activation of compensatory pathways remain a challenge in the treatment of cancer patients, revealing the need to explore alternative strategies to the classical anti-angiogenic drugs. In this review, we will describe some alternative strategies to inhibit tumor vascularization, including targeting of antigens and signaling pathways overexpressed by tumor endothelial cells, the development of endothelial vaccinations, and the use of extracellular vesicles. In addition, anti-angiogenic drugs with normalizing effects on tumor vessels will be discussed. Finally, we will present the concept of endothelial demesenchymalization as an alternative approach to restore normal endothelial cell phenotype.

Tumor microenvironment is particularly enriched with extracellular ATP (eATP), but conflicting evidence has been provided on its functional effects on tumor growth and vascular remodeling. We have previously shown that high eATP concentrations exert a strong anti-migratory, antiangiogenic and normalizing activity on human tumor-derived endothelial cells (TECs). Since both metabotropic and ionotropic purinergic receptors trigger cytosolic calcium increase ([Ca2+]c), the present work investigated the properties of [Ca2+]c events elicited by high eATP in TECs and their role in anti-migratory activity. In particular, the quantitative and kinetic properties of purinergic-induced Ca2+ release from intracellular stores and Ca2+ entry from extracellular medium were investigated. The main conclusions are: (1) stimulation of TECs with high eATP triggers [Ca2+]c signals which include Ca2+ mobilization from intracellular stores (mainly ER) and Ca2+ entry through the plasma membrane, (2) the long-lasting Ca2+ influx phase requires both store-operated Ca2+ entry (SOCE) and non-SOCE components, (3) SOCE is not significantly involved in the antimigratory effect of high ATP stimulation, (4) ER is the main source for intracellular Ca2+ release by eATP: it is required for the constitutive migratory potential of TECs but is not the only determinant for the inhibitory effect of high eATP, (5) a complex interplay occurs among ER, mitochondria and lysosomes upon purinergic stimulation, (6) high eUTP is unable to inhibit TEC migration and evokes [Ca2+]c signals very similar to those described for eATP. The potential role played by store-independent Ca2+ entry and Ca2+-independent events in the regulation of TEC migration by high purinergic stimula deserves future investigation.

To clarify the mechanisms of interaction between SiONontoxic doses of SiOTRPV4, Cx and Panx-like channels are the major components of the NP-induced inward currents. Preincubation with the antioxidant N-acetyl-L-cysteine strongly reduced the [CaThese findings suggest that SiO

Ethnopharmacological relevance Several traditional medicinal herbs are widely used for dermatologic and cosmetic preparations. The beneficial skin repair activity is detected in various phases of wound-healing process, such as cell-cell, cell-matrix interactions or collagen synthesis. Aim Of The Study: The study assessed the effects of Opuntia ficus-indica (L.) Mill. (Opuntia) and Milk Thistle (MT) (Silybum marianum (L.) Gaerth) on adult keratinocytes (HaCaT) functioning under basal condition or in the presence of mechanical damage (wounded cells). Materials and methods The role of the natural compounds was tested on HaCaT grown in mono-culture and tri-culture configurations. In tri-cultures models, HaCaT were treated with the conditioned media (CM) obtained by Human Normal Dermal Fibroblast (NHDF) and Human Dermal Microvascular Endothelial cells (HMVEC) co-cultures. Specifically, were tested cell viability, oxidative stress mechanisms (cytokines release and lipid peroxidation) and cellular remodelling (growth factors release or metalloproteinase modulation). Moreover, the migratory potential of HaCaT was analysed by the use of wound healing in vitro assay. Results Opuntia and MT differently modified the metabolism (EGF, MMP-9), and the migratory properties of HaCaT both under physiological conditions or upon mechanical damage (wounded cells). Moreover, both compounds modulated HaCaT response to oxidative stress. The response to the natural compounds were modified, and in some cases potentiated, in tri-culture configuration systems. Conclusions The data demonstrated that in vitro tri-culture approach is suitable to characterize the role of natural compounds on the complex communication between dermal-epidermal cellular components and microvascular endothelium. Specifically, Opuntia and MT are good alternatives to synthetic compounds in skin repair promotion.

The human cytomegalovirus (HCMV) US12 gene family comprises a set of 10 contiguous genes (US12 to US21) with emerging roles in the regulation of virus cell tropism, virion composition, and immunoevasion. Of all of the US12 gene products, pUS21 shows the highest level of identity with two cellular transmembrane BAX inhibitor motif-containing (TMBIM) proteins: Bax inhibitor-1 and Golgi anti-apoptotic protein, both of which are involved in the regulation of cellular Ca(2+) homeostasis and adaptive cell responses to stress conditions. Here, we report the US21 protein to be a viral-encoded ion channel that regulates intracellular Ca(2+) homeostasis and protects cells against apoptosis. Indeed, we show pUS21 to be a 7TMD protein expressed with late kinetics that accumulates in ER-derived vesicles. Deletion or inactivation of the US21 gene resulted in reduced HCMV growth, even in fibroblasts, due to reduced gene expression. Ratiometric fluorescence imaging assays revealed that expression of pUS21 reduces the Ca(2+) content of intracellular ER stores. An increase in cell resistance to intrinsic apoptosis was then observed as an important cytobiological consequence of the pUS21-mediated alteration of intracellular Ca(2+) homeostasis. Moreover, a single point mutation in the putative pore of pUS21 impaired the reduction of ER Ca(2+) concentration and attenuated the antiapoptotic activity of pUS21wt, supporting a functional link with its ability to manipulate Ca(2+) homeostasis. Together, these results suggest pUS21 of HCMV constitutes a TMBIM-derived viroporin that may contribute to HCMV’s overall strategy to counteract apoptosis in infected cells.

Mesenchymal stem cells (MSCs) can be harvested from different sites in the oral cavity, representing a reservoir of cells useful for regenerative purposes. As direct comparisons between at least two types of MSCs deriving from the same patient are surprisingly rare in scientific literature, we isolated and investigated the osteoinductive potential of dental pulp stem cells (DPSCs) and buccal fat pad stem cells (BFPSCs). MSCs were isolated from the third molar dental pulp and buccal fat pads of 12 patients. The number of viable cells was quantified through manual count. Proliferation and osteodifferentiation assays, flow cytometry analysis of cell phenotypes, and osteocalcin release in vitro were performed. The isolation of BFPSCs and DPSCs was successful in 7 out of 12 (58%) and 3 out of 12 (25%) of retrieved samples, respectively. The yield of cells expressing typical stem cell markers and the level of proliferation were higher in BFPSCs than in DPSCs. Both BFP-SCs and DPSCs differentiated into osteoblast-like cells and were able to release a mineralized matrix. The release of osteocalcin, albeit greater for BFPSCs, did not show any significant difference between BFPSCs and DPSCs. The yield of MSCs depends on their site of origin as well as on the protocol adopted for their isolation. Our data show that BFP is a valuable source for the derivation of MSCs that can be used for regenerative treatments.

The biological importance of circulatory blood supply and angiogenesis for hair growth is now well recognized, but the their regulatory mechanisms require more mechanistic investigation. In vitro cocultures and tricultures can be successfully employed to greatly improve our knowledge on paracrine crosstalk between cell types that populate the dermal-epidermal interface and cutaneous vasculature. Here we report that human dermal fibroblasts (NHDF) promote viability and proliferation of microvascular endothelial cells (HMVEC), while HMVEC are not mitogenic for NHDF. In triculture setup, conditioned media (CM) obtained by cocultures (HMVEC/NHDF or HMVEC/follicle fibroblasts) differently modulate growth and proliferation of keratinocytes and alter the expression of metabolic and pro-inflammatory markers. In conclusion, tricultures were successfully employed to characterize in vitro dermal-epithelial and endothelial interactions and could integrate ex vivo and in vivo approaches by the use of high-throughput and standardized protocols in controlled conditions. J. Cell. Physiol. 232: 897-903, 2017. © 2016 Wiley Periodicals, Inc.

Silicon nitride (Si3N4) has been introduced clinically as an orthopedic biomaterial for interbody fusion devices and in joint replacements. However, the production of complex shapes through conventional mechanical machining is difficult and expensive and limits interesting applications. Thus, several electrically conductive reinforcements to the Si3N4 matrix, like TiN, have been proposed, generating composites suitable to be wrought by electrical discharge machining (EDM). In this study, Si3N4-TiN with high strength, low density, and good electric conductivity wrought by EDM was studied. The role of surface finishing was investigated comparing the interface generated during the EDM process to that resulting from further polishing. The different topographical features were assessed by electron microscopy, energy dispersive X-ray spectrometry, and profilometry. Surface wettability was also determined based on the measurement of the OCA of water and diiodomethane. The biological responses induced in MC3T3 cells, a widely diffused osteoblast model, were correlated with the surface pattern. The unpolished samples could promote better cell viability, with a more relevant effect on the cytoskeleton arrangement as highlighted by numerous cytoplasmic extensions and filopodia-like structures and the high number of focal adhesions, while MC3T3 cells grown on polished Si3N4-TiN specimens displayed a flat morphology. In addition, the unpolished Si3N4-TiN increased osteocalcin production and calcium deposition. Taken together, these data support the biocompatibility and in vitro osteogenic properties of the electroconductive Si3N4-TiN investigated. Further in vivo studies are required to explore the possible use of bone implants directly obtained by EDM. © 2016, Springer Science+Business Media New York.

Some natural compounds, including flavonoids, are active in vasculature re-growth during hair follicle disruption, but their effects have not been yet evaluated directly on microvascular endothelial cells. Skin vascularisation regulates the physiological blood supply required for hair growth and its dysregulation is the basis of several human diseases. Follicle-derived vascular endothelial growth factor (VEGF) release from follicular keratinocytes promotes perifollicular vascularisation and increases follicle and hair size, while blockade of VEGF-mediated angiogenesis leads to impaired hair growth. Here, we tested three flavonoids, namely visnadin (VSD), hesperidin (HSP) and baicalin (BC), on cultured human microvascular endothelial cells (HMEC), comparing their effects with minoxidil (MXD), a synthetic drug broadly used in the treatment of androgenetic alopecia. The response to these compounds was assayed in terms of endothelial survival, proliferation, tubulogenesis and proangiogenic signalling. We show that BC promotes HMEC proliferation, while both VSD and MXD enhance tubulogenesis. Interestingly, only HSP increases VEGFR-2 phosphorylation.

This paper aimed to assess how four different and representative dental implant surfaces (machined MAC, grit blasted GB, acid etched AE and grit blasted acid etched GBAE) could affect cell adhesion possibly modulating protein adsorption and a paramount cell receptor such as α5s1 integrin. Based on non-contact 3D profilometry, GBAE was rougher than the other surfaces, while GB and AE reached similar intermediate Sa values and MAC resulted the smoothest one. According to X-ray Photoelectron Spectroscopy and Raman spectroscopy, all the surfaces showed amorphous titania with similar amounts of carbon contaminants. The lesser protein adsorption and delayed cell adhesion of GB, which did not hinder cell proliferation, were correlated to the altered dispersive to polar SE ratio, that was at least double-fold for GB (2.6) compared to MAC (0.8), SL (1.3) and AE (1.2). The biological response in vitro relied on β1 integrin activation that cooperated with the putative mechano-protein transient receptor potential vanilloid 4 (TRPV4) in determining the adhesion of the osteoblasts. In fact, it could be rescued by over activating β1 integrin. On the other hand, silencing TRPV4 strongly inhibited cell adhesion on selected substrates, proving the role of this protein in mediating osteoblasts adhesion on titanium substrates.