Combining antibiotic-loaded bone cement-based free vastus lateralis muscle-sparing flap with split-thickness skin grafts: A reliable strategy for reconstructing diabetic foot ulcers at non-weight-bearing areas.

Diabetic foot ulcers (DFUs) present significant challenges due to their associated amputation rates, mortality, treatment complexity and excessive costs. Our earlier work introduced a wound surgical integrated treatment (WSIT) for DFUs, yielding promising outcomes. This study focuses on a specific WSIT protocol employing antibiotic-loaded bone cement (ALBC) in the first Stage, and free vastus lateralis muscle-sparing (VLMS) flaps and split-thickness skin grafts (STSGs) in the second stage to repair non-weight-bearing DFUs. From July 2021 to July 2023, seven DFU patients (aged 47-71 years) underwent this treatment. Demographic data, hospital stay and repair surgery times were collected. Histological and immunohistochemical analyses assessed angiogenesis, collagen deposition and inflammation. SF-36 questionnaire measured pre- and postoperative quality of life. Preoperative ultrasound Doppler showed that the peak blood flow velocity of the recipient area artery was significantly >30 cm/s (38.6 ± 6.8 cm/s) in all patients. Muscle flap sizes varied from 8 × 3.5 × 1 to 18 × 6 × 2 cm. The operation time of the repair surgery was 156.9 ± 15.08 minutes, and the hospital stay was 18.9 ± 3.3 days. Histological analysis proved that covering DFUs with ALBC induced membrane formation and increased collagen, neovascularization and M2 macrophages fraction while reducing M1 macrophages one. All grafts survived without amputation during a 7- to 24-month follow-up, during which SF-36 scores significantly improved. A combination of ALBC with free VLMS flaps and STSGs proved to be safe and effective for reconstructing non-weight-bearing DFUs. It rapidly controlled infection, enhanced life quality and foot function, and reduced hospitalization time. We advocate integrating this strategy into DFU treatment plans.

Human Keratinocytes and Fibroblasts Co-Cultured on Silk Fibroin Scaffolds Exosomally Overrelease Angiogenic and Growth Factors.

Objectives: The optimal healing of skin wounds, deep burns, and chronic ulcers is an important clinical problem. Attempts to solve it have been driving the search for skin equivalents based on synthetic or natural polymers. Methods: Consistent with this endeavor, we used regenerated silk fibroin (SF) from Bombyx mori to produce a novel compound scaffold by welding a 3D carded/hydroentangled SF-microfiber-based nonwoven layer (C/H-3D-SFnw; to support dermis engineering) to an electrospun 2D SF nanofiber layer (ESFN; a basal lamina surrogate). Next, we assessed-via scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, mono- and co-cultures of HaCaT keratinocytes and adult human dermal fibroblasts (HDFs), dsDNA assays, exosome isolation, double-antibody arrays, and angiogenesis assays-whether the C/H-3D-SFnws/ESFNs would allow the reconstitution of a functional human skin analog in vitro. Results: Physical analyses proved that the C/H-3D-SFnws/ESFNs met the requirements for human soft-tissue-like implants. dsDNA assays revealed that co-cultures of HaCaTs (on the 2D ESFN surface) and HDFs (inside the 3D C/H-3D-SFnws) grew more intensely than did the respective monocultures. Double-antibody arrays showed that the CD9+/CD81+ exosomes isolated from the 14-day pooled growth media of HDF and/or HaCaT mono- or co-cultures conveyed 35 distinct angiogenic/growth factors (AGFs). However, versus monocultures' exosomes, HaCaT/HDF co-cultures' exosomes (i) transported larger amounts of 15 AGFs, i.e., PIGF, ANGPT-1, bFGF, Tie-2, Angiogenin, VEGF-A, VEGF-D, TIMP-1/-2, GRO-α/-ß/-γ, IL-1ß, IL-6, IL-8, MMP-9, and MCP-1, and (ii) significantly more strongly stimulated human dermal microvascular endothelial cells to migrate and assemble tubes/nodes in vitro. Conclusions: Our results showed that both cell-cell and cell-SF interactions boosted the exosomal release of AGFs from HaCaTs/HDFs co-cultured on C/H-3D-SFnws/ESFNs. Hence, such exosomes are an asset for prospective clinical applications as they advance cell growth and neoangiogenesis and consequently graft take and skin healing. Moreover, this new integument analog could be instrumental in preclinical and translational studies on human skin pathophysiology and regeneration.

NLRP3 Inflammasome's Activation in Acute and Chronic Brain Diseases-An Update on Pathogenetic Mechanisms and Therapeutic Perspectives with Respect to Other Inflammasomes.

Increasingly prevalent acute and chronic human brain diseases are scourges for the elderly. Besides the lack of therapies, these ailments share a neuroinflammation that is triggered/sustained by different innate immunity-related protein oligomers called inflammasomes. Relevant neuroinflammation players such as microglia/monocytes typically exhibit a strong NLRP3 inflammasome activation. Hence the idea that NLRP3 suppression might solve neurodegenerative ailments. Here we review the recent Literature about this topic. First, we update conditions and mechanisms, including RNAs, extracellular vesicles/exosomes, endogenous compounds, and ethnic/pharmacological agents/extracts regulating NLRP3 function. Second, we pinpoint NLRP3-activating mechanisms and known NLRP3 inhibition effects in acute (ischemia, stroke, hemorrhage), chronic (Alzheimer's disease, Parkinson's disease, Huntington's disease, MS, ALS), and virus-induced (Zika, SARS-CoV-2, and others) human brain diseases. The available data show that (i) disease-specific divergent mechanisms activate the (mainly animal) brains NLRP3; (ii) no evidence proves that NLRP3 inhibition modifies human brain diseases (yet ad hoc trials are ongoing); and (iii) no findings exclude that concurrently activated other-than-NLRP3 inflammasomes might functionally replace the inhibited NLRP3. Finally, we highlight that among the causes of the persistent lack of therapies are the species difference problem in disease models and a preference for symptomatic over etiologic therapeutic approaches. Therefore, we posit that human neural cell-based disease models could drive etiological, pathogenetic, and therapeutic advances, including NLRP3's and other inflammasomes' regulation, while minimizing failure risks in candidate drug trials.

"Other Than NLRP3" Inflammasomes: Multiple Roles in Brain Disease.

Human neuroinflammatory and neurodegenerative diseases, whose prevalence keeps rising, are still unsolved pathobiological/therapeutical problems. Among others, recent etiology hypotheses stressed as their main driver a chronic neuroinflammation, which is mediated by innate immunity-related protein oligomers: the inflammasomes. A panoply of exogenous and/or endogenous harmful agents activates inflammasomes' assembly, signaling, and IL-1ß/IL-18 production and neural cells' pyroptotic death. The underlying concept is that inflammasomes' chronic activation advances neurodegeneration while their short-lasting operation restores tissue homeostasis. Hence, from a therapeutic standpoint, it is crucial to understand inflammasomes' regulatory mechanisms. About this, a deluge of recent studies focused on the NLRP3 inflammasome with suggestions that its pharmacologic block would hinder neurodegeneration. Yet hitherto no evidence proves this view. Moreover, known inflammasomes are numerous, and the mechanisms regulating their expression and function may vary with the involved animal species and strains, as well as organs and cells, and the harmful factors triggered as a result. Therefore, while presently leaving out some little-studied inflammasomes, this review focuses on the "other than NLRP3" inflammasomes that participate in neuroinflammation's complex mechanisms: NLRP1, NLRP2, NLRC4, and AIM2. Although human-specific data about them are relatively scant, we stress that only a holistic view including several human brain inflammasomes and other potential pathogenetic drivers will lead to successful therapies for neuroinflammatory and neurodegenerative diseases.

Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction.

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm-1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 µmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 µmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Adult Human Vascular Smooth Muscle Cells on 3D Silk Fibroin Nonwovens Release Exosomes Enriched in Angiogenic and Growth-Promoting Factors.

BACKGROUND: Our earlier works showed the quick vascularization of mouse skin grafted Bombyx mori 3D silk fibroin nonwoven scaffolds (3D-SFnws) and the release of exosomes enriched in angiogenic/growth factors (AGFs) from in vitro 3D-SFnws-stuck human dermal fibroblasts (HDFs). Here, we explored whether coronary artery adult human smooth muscle cells (AHSMCs) also release AGFs-enriched exosomes when cultured on 3D-SFnws in vitro. METHODS: Media with exosome-depleted FBS served for AHSMCs and human endothelial cells (HECs) cultures on 3D-SFnws or polystyrene. Biochemical methods and double-antibody arrays assessed cell growth, metabolism, and intracellular TGF-ß and NF-κB signalling pathways activation. AGFs conveyed by CD9+/CD81+ exosomes released from AHSMCs were double-antibody array analysed and their angiogenic power evaluated on HECs in vitro. RESULTS: AHSMCs grew and consumed D-glucose more intensely and showed a stronger phosphorylation/activation of TAK-1, SMAD-1/-2/-4/-5, ATF-2, c-JUN, ATM, CREB, and an IκBα phosphorylation/inactivation on SFnws vs. polystyrene, consistent overall with a proliferative/secretory phenotype. SFnws-stuck AHSMCs also released exosomes richer in IL-1α/-2/-4/-6/-8; bFGF; GM-CSF; and GRO-α/-ß/-γ, which strongly stimulated HECs' growth, migration, and tubes/nodes assembly in vitro. CONCLUSIONS: Altogether, the intensified AGFs exosomal release from 3D-SFnws-attached AHSMCs and HDFs could advance grafts' colonization, vascularization, and take in vivo-noteworthy assets for prospective clinical applications.

Danger-Sensing/Patten Recognition Receptors and Neuroinflammation in Alzheimer's Disease.

Fibrillar aggregates and soluble oligomers of both Amyloid-ß peptides (Aßs) and hyperphosphorylated Tau proteins (p-Tau-es), as well as a chronic neuroinflammation are the main drivers causing progressive neuronal losses and dementia in Alzheimer's disease (AD). However, the underlying pathogenetic mechanisms are still much disputed. Several endogenous neurotoxic ligands, including Aßs, and/or p-Tau-es activate innate immunity-related danger-sensing/pattern recognition receptors (PPRs) thereby advancing AD's neuroinflammation and progression. The major PRR families involved include scavenger, Toll-like, NOD-like, AIM2-like, RIG-like, and CLEC-2 receptors, plus the calcium-sensing receptor (CaSR). This quite intricate picture stresses the need to identify the pathogenetically topmost Aß-activated PRR, whose signaling would trigger AD's three main drivers and their intra-brain spread. In theory, the candidate might belong to any PRR family. However, results of preclinical studies using in vitro nontumorigenic human cortical neurons and astrocytes and in vivo AD-model animals have started converging on the CaSR as the pathogenetically upmost PRR candidate. In fact, the CaSR binds both Ca2+ and Aßs and promotes the spread of both Ca2+ dyshomeostasis and AD's three main drivers, causing a progressive neurons' death. Since CaSR's negative allosteric modulators block all these effects, CaSR's candidacy for topmost pathogenetic PRR has assumed a growing therapeutic potential worth clinical testing.

CaSR Antagonist (Calcilytic) NPS 2143 Hinders the Release of Neuroinflammatory IL-6, Soluble ICAM-1, RANTES, and MCP-2 from Aß-Exposed Human Cortical Astrocytes.

Available evidence shows that human cortical neurons' and astrocytes' calcium-sensing receptors (CaSRs) bind Amyloid-beta (Aß) oligomers triggering the overproduction/oversecretion of several Alzheimer's disease (AD) neurotoxinseffects calcilytics suppress. We asked whether AßCaSR signaling might also play a direct pro-neuroinflammatory role in AD. Cortical nontumorigenic adult human astrocytes (NAHAs) in vitro were untreated (controls) or treated with Aß25-35±ï€ NPS 2143 (a calcilytic) and any proinflammatory agent in their protein lysates and growth media assayed via antibody arrays, enzyme-linked immunosorbent assays (ELISAs), and immunoblots. Results show Aß•CaSR signaling upregulated the synthesis and release/shedding of proinflammatory interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1) (holoprotein and soluble [s] fragment), Regulated upon Activation, normal T cell Expressed and presumably Secreted (RANTES), and monocyte chemotactic protein (MCP)-2. Adding NPS 2143 (i) totally suppressed IL-6's oversecretion while remarkably reducing the other agents' over-release; and (ii) more effectively than Aß alone increased over controls the four agents' distinctive intracellular accumulation. Conversely, NPS 2143 did not alter Aß-induced surges in IL-1ß, IL-3, IL-8, and IL-16 secretion, consequently revealing their Aß•CaSR signaling-independence. Finally, Aß25-35±ï€ NPS 2143 treatments left unchanged MCP-1's and TIMP-2's basal expression. Thus, NAHAs Aß•CaSR signaling drove four proinflammatory agents' over-release that NPS 2143 curtailed. Therefore, calcilytics would also abate NAHAs' Aß•CaSR signaling direct impact on AD's neuroinflammation.

Maladaptive remodeling of pulmonary artery root autografts after Ross procedure: A proteomic study.

OBJECTIVE: Pulmonary autograft root dilatation is the major long-term complication after Ross procedure and the leading cause for reoperation. However, the mechanisms underlying dilatation remain to be elucidated. This study analyzed the proteomic changes seen in the dilated pulmonary autograft compared with normal pulmonary artery and aorta tissues. METHODS: Pulmonary autograft surgical samples were taken from 9 consecutive patients (mean age 37 ± 14; 15-51 years) with mean diameters of 5.2 ± 0.5 cm (4.6-5.8 cm) reoperated 8 to 16 years after Ross procedure. Control pulmonary artery and aorta samples were from 7 age- and sex-matched cardiac donors. Tunicae mediae from all samples were processed for proteomic analysis via 2-dimensional electrophoresis, matrix-assisted-laser-desorption-ionization-time of flight/mass spectrometry, and bioinformatics. The thus-identified putatively relevant proteins were validated via Western immunoblotting. RESULTS: Pulmonary autograft proteome features differed markedly from control pulmonary arteries, since proteins related to focal adhesions (eg, paxillin), cytoskeleton (eg, vimentin), and metalloprotease-regulating proteoglycans (eg, testican-2) were significantly up-regulated, whereas significant decreases occurred in microfibril-associated glycoprotein1, which controls elastic fiber buildup. Profound changes also occurred in cell-signaling proteins, ie, increases in soluble Jagged-1 fragment and ectodysplasin-2 receptor, and decreases in Notch-1 intracellular domain fragment. Moreover, pulmonary autograft expression levels of Paxillin, Vimentin, Jagged-1 fragment, and Notch1 intracellular domain fragment also differed from those of control aorta. CONCLUSIONS: This study provides the first description of the specific proteomic features of dilated pulmonary autograft tunica media, which separate them sharply not only from those of control pulmonary artery and aorta but also of aortic aneurysms. These findings suggest that dilated pulmonary autografts undergo a unique maladaptive remodeling process deserving further investigation.

Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests.

Silk fibroin (SF) is an eligible biomaterial for the development of small caliber vascular grafts for substitution, repair, and regeneration of blood vessels. This study presents the properties of a newly designed multi-layered SF tubular scaffold for vascular grafting (SilkGraf). The wall architecture consists of two electrospun layers (inner and outer) and an intermediate textile layer. The latter was designed to confer high mechanical performance and resistance on the device, while electrospun layers allow enhancing its biomimicry properties and host's tissues integration. In vitro cell interaction studies performed with adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) demonstrated that the electrospun layers favor cell adhesion, survival, and growth. Once cultured in vitro on the SF scaffold the three cell types showed an active metabolism (consumption of glucose and glutamine, release of lactate), and proliferation for up to 20 days. HAAF cells grown on SF showed a significantly lower synthesis of type I procollagen than on polystyrene, meaning a lower fibrotic effect of the SF substrate. The cytokine and chemokine expression patterns were investigated to evaluate the cells' proliferative and pro-inflammatory attitude. Interestingly, no significant amounts of truly pro-inflammatory cytokines were secreted by any of the three cell types which exhibited a clearly proliferative profile. Good hemocompatibility was observed by complement activation, hemolysis, and hematology assays. Finally, the results of an in vivo preliminary pilot trial on minipig and sheep to assess the functional behavior of implanted SF-based vascular graft identified the sheep as the more apt animal model for next medium-to-long term preclinical trials.

Family C G-Protein-Coupled Receptors in Alzheimer's Disease and Therapeutic Implications.

Alzheimer's disease (AD), particularly its sporadic or late-onset form (SAD/LOAD), is the most prevalent (96-98% of cases) neurodegenerative dementia in aged people. AD's neuropathology hallmarks are intrabrain accumulation of amyloid-ß peptides (Aßs) and of hyperphosphorylated Tau (p-Tau) proteins, diffuse neuroinflammation, and progressive death of neurons and oligodendrocytes. Mounting evidences suggest that family C G-protein-coupled receptors (GPCRs), which include γ-aminobutyric acid B receptors (GABABRs), metabotropic glutamate receptors (mGluR1-8), and the calcium-sensing receptor (CaSR), are involved in many neurotransmitter systems that dysfunction in AD. This review updates the available knowledge about the roles of GPCRs, particularly but not exclusively those expressed by brain astrocytes, in SAD/LOAD onset and progression, taking stock of their respective mechanisms of action and of their potential as anti-AD therapeutic targets. In particular, GABABRs prevent Aßs synthesis and neuronal hyperexcitability and group I mGluRs play important pathogenetic roles in transgenic AD-model animals. Moreover, the specific binding of Aßs to the CaSRs of human cortical astrocytes and neurons cultured in vitro engenders a pathological signaling that crucially promotes the surplus synthesis and release of Aßs and hyperphosphorylated Tau proteins, and also of nitric oxide, vascular endothelial growth factor-A, and proinflammatory agents. Concurrently, Aßs•CaSR signaling hinders the release of soluble (s)APP-α peptide, a neurotrophic agent and GABABR1a agonist. Altogether these effects progressively kill human cortical neurons in vitro and likely also in vivo. Several CaSR's negative allosteric modulators suppress all the noxious effects elicited by Aßs•CaSR signaling in human cortical astrocytes and neurons thus safeguarding neurons' viability in vitro and raising hopes about their potential therapeutic benefits in AD patients. Further basic and clinical investigations on these hot topics are needed taking always heed that activation of the several brain family C GPCRs may elicit divergent upshots according to the models studied.

Over Expressed TKTL1, CIP-2A, and B-MYB Proteins in Uterine Cervix Epithelium Scrapings as Potential Risk Predictive Biomarkers in HR-HPV-Infected LSIL/ASCUS Patients.

High oncogenic risk human papillomaviruses (HR-HPVs) promote cervical carcinoma development, the fourth most common feminine cancer. A slow oncodevelopmental phase-defined histopathologically as Cervical Intraepithelial Neoplasia (CIN) grades 1-3, or cytologically as Low- or High-grade Squamous Intraepithelial Lesions (LSIL or HSIL)-precedes the malignancy. Cervical carcinoma screenings through HR-HPV genotyping and Pap smears are regularly performed in Western countries. Faulty cytology screening or genotyping or patients' non-compliance with follow-ups can let slip an oncoprogression diagnosis. Novel biomarker tests flanking HR-HPV genotyping and cytology could objectively predict the risk of disease progression thus helping triage LSIL/ASCUS patients. Here, anonymized leftovers of fresh cervical epithelium scrapings from twice (LSIL/ASCUS and HR-HPV DNA)-positive and twice (Pap smear- and HR-HPV DNA)-negative (control) patients in a proteome-preserving solution served to assess the biomarker worth of three cervical carcinoma-related proteins, i.e., B-MYB (or MYBL2), Cancerous Inhibitor of PP2A (CIP-2a), and transketolase-like1 (TKTL1). Leftovers anonymity was strictly kept and storage at -80°C, protein extraction, immunoblotting, and band densitometry were blindly performed. Only after tests completion, the anonymous yet code-corresponding HR-HPV-genotyping and cytology data allowed to assign each sample to the twice-positive or twice-negative group. Descriptive statistics showed that the three proteins levels significantly increased in the twice-positive vs. twice-negative scrapings. Diagnostic ROC curve analysis identified each protein's Optimal Decision Threshold (OTD) showing that TKTL1 and CIP-2a are stronger risk predictive biomarkers (Sensitivity, 0.91-0.93; Specificity, 0.77-0.83) than B-MYB. Logistic Regression coupled with Likelihood-Ratio Tests confirmed that a highly significant relation links increasing TKTL1/CIP-2a/B-MYB protein levels in twice-positive cervical scrapings to the risk of HR-HPV-driven oncoprogression. Finally, a 3 year clinical follow-up showed that 13 patients (50% of total) of the twice-positive group with biomarker values over OTDs compliantly underwent scheduled colposcopy and biopsy. Of these, 11 (i.e., 84.7%) received a positive histological diagnosis, i.e., CIN1 (n = 5; 38.5%) or CIN2/CIN2+ (n = 6; 46,2%). Therefore, TKTL1/CIP-2a/B-MYB protein levels could objectively predict oncoprogression risk in twice (HR-HPV- and Pap smear)-positive women. Further studies will assess the translatability of these findings into clinical settings.

Studies on sporadic non-syndromic thoracic aortic aneurysms: 1. Deregulation of Jagged/Notch 1 homeostasis and selection of synthetic/secretor phenotype smooth muscle cells.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. The study aimed at defining the peculiar morphologic and molecular changes occurring in the media layer of SNSTAAs. Design This study was based on a single centre design. Methods Media layer samples taken from seven carefully selected SNSTAAs and seven reference patients (controls) were investigated via quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, quantitative histology, and immunohistochemistry/immunofluorescence. Results In SNSTAAs media, aortic smooth muscle cells numbers were halved due to an apoptotic process coupled with a negligible cell proliferation. Cystathionine γ-lyase was diffusely up-regulated. Surviving aortic smooth muscle cells exhibited diverging phenotypes: in inner- and outer-media contractile cells prevailed, having higher contents of smooth-muscle-α-actin holoprotein (45-kDa) and of caspase-3-cleaved smooth-muscle-α-actin 25-kDa fragments; in mid-media, aortic smooth muscle cells exhibited a synthetic/secretor phenotype, down-regulating vimentin, but up-regulating glial fibrillary acidic protein, trans-Golgi network 46 protein, Jagged1 (172-kDa) holoprotein, and Jagged1's receptor Notch1. Extracellular soluble Jagged1 (42-kDa) fragments accumulated. Conclusions In SNSTAAs, there is a relentless aortic smooth muscle cells attrition caused by the up-regulated cystathionine γ-lyase. In mid-media, synthetic/secretor aortic smooth muscle cells intensify Jagged1/NOTCH1 signalling in the attempt to counterbalance the weakened aortic wall, due to aortic smooth muscle cells net loss and mechanical stress. Synthetic/secretor aortic smooth muscle cells are apoptosis-prone, and the accruing thrombin-cleaved Jagged1 fragments counteract the otherwise useful effects of Jagged1/NOTCH1 signalling, thus hampering tissue homeostasis/remodelling, and aortic smooth muscle cells adhesion, differentiation, and migration.

Studies on sporadic non-syndromic thoracic aortic aneurysms: II. Alterations of extra-cellular matrix components and focal adhesion proteins.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.

Amyloid ß-Exposed Human Astrocytes Overproduce Phospho-Tau and Overrelease It within Exosomes, Effects Suppressed by Calcilytic NPS 2143-Further Implications for Alzheimer's Therapy.

The two main drivers of Alzheimer's disease (AD), amyloid-ß (Aß) and hyperphosphorylated Tau (p-Tau) oligomers, cooperatively accelerate AD progression, but a hot debate is still ongoing about which of the two appears first. Here we present preliminary evidence showing that Tau and p-Tau are expressed by untransformed cortical adult human astrocytes in culture and that exposure of such cells to an Aß42 proxy, Aß25-35, which binds the calcium-sensing receptor (CaSR) and activates its signaling, significantly increases intracellular p-Tau levels, an effect CaSR antagonist (calcilytic) NPS 2143 wholly hinders. The astrocytes also release both Tau and p-Tau by means of exosomes into the extracellular medium, an activity that could mediate p-Tau diffusion within the brain. Preliminary data also indicate that exosomal levels of p-Tau increase after Aß25-35 exposure, but remain unchanged in cells pre-treated for 30-min with NPS 2143 before adding Aß25-35. Thus, our previous and present findings raise the unifying prospect that Aß•CaSR signaling plays a crucial role in AD development and progression by simultaneously activating (i) the amyloidogenic processing of amyloid precursor holoprotein, whose upshot is a surplus production and secretion of Aß42 oligomers, and (ii) the GSK-3ß-mediated increased production of p-Tau oligomers which are next released extracellularly inside exosomes. Therefore, as calcilytics suppress both effects on Aß42 and p-Tau metabolic handling, these highly selective antagonists of pathological Aß•CaSR signaling would effectively halt AD's progressive spread preserving patients' cognition and life quality.

Calcium-Sensing Receptor Antagonist NPS 2143 Restores Amyloid Precursor Protein Physiological Non-Amyloidogenic Processing in Aß-Exposed Adult Human Astrocytes.

Physiological non-amyloidogenic processing (NAP) of amyloid precursor holoprotein (hAPP) by α-secretases (e.g., ADAM10) extracellularly sheds neurotrophic/neuroprotective soluble (s)APPα and precludes amyloid-ß peptides (Aßs) production via ß-secretase amyloidogenic processing (AP). Evidence exists that Aßs interact with calcium-sensing receptors (CaSRs) in human astrocytes and neurons, driving the overrelease of toxic Aß42/Aß42-os (oligomers), which is completely blocked by CaSR antagonist (calcilytic) NPS 2143. Here, we investigated the mechanisms underlying NPS 2143 beneficial effects in human astrocytes. Moreover, because Alzheimer's disease (AD) involves neuroinflammation, we examined whether NPS 2143 remained beneficial when both fibrillary (f)Aß25-35 and a microglial cytokine mixture (CMT) were present. Thus, hAPP NAP prevailed over AP in untreated astrocytes, which extracellularly shed all synthesized sAPPα while secreting basal Aß40/42 amounts. Conversely, fAß25-35 alone dramatically reduced sAPPα extracellular shedding while driving Aß42/Aß42-os oversecretion that CMT accelerated but not increased, despite a concurring hAPP overexpression. NPS 2143 promoted hAPP and ADAM10 translocation to the plasma membrane, thereby restoring sAPPα extracellular shedding and fully suppressing any Aß42/Aß42-os oversecretion, but left hAPP expression unaffected. Therefore, as anti-AD therapeutics calcilytics support neuronal viability by safeguarding astrocytes neurotrophic/neuroprotective sAPPα shedding, suppressing neurons and astrocytes Aß42/Aß42-os build-up/secretion, and remaining effective even under AD-typical neuroinflammatory conditions.

Increased Calcium-Sensing Receptor Immunoreactivity in the Hippocampus of a Triple Transgenic Mouse Model of Alzheimer's Disease.

The Calcium-Sensing Receptor (CaSR) is a G-protein coupled, 7-transmembrane domain receptor ubiquitously expressed throughout the body, brain including. The role of CaSR in the CNS is not well understood; its expression is increasing during development, which has been implicated in memory formation and consolidation, and CaSR localization in nerve terminals has been related to synaptic plasticity and neurotransmission. There is an emerging evidence of CaSR involvement in neurodegenerative disorders and Alzheimer's disease (AD) in particular, where the over-production of ß-amyloid peptides was reported to activate CaSR. In the present study, we performed CaSR immunohistochemical and densitometry analysis in the triple transgenic mouse model of AD (3xTg-AD). We found an increase in the expression of CaSR in hippocampal CA1 area and in dentate gyrus in the 3xTg-AD mice when compared to non-transgenic control animals. This increase was significant at 9 months of age and further increased at 12 and 18 months of age. This increase paralleled the accumulation of ß-amyloid plaques with age. Increased expression of CaSR favors ß-amyloidogenic pathway following direct interactions between ß-amyloid and CaSR and hence may contribute to the pathological evolution of the AD. In the framework of this paradigm CaSR may represent a novel therapeutic target.

Biocompatible Silk Noil-Based Three-Dimensional Carded-Needled Nonwoven Scaffolds Guide the Engineering of Novel Skin Connective Tissue.

Retracting hypertrophic scars resulting from healed burn wounds heavily impact on the patients' life quality. Biomaterial scaffolds guiding burned-out skin regeneration could suppress or lessen scar retraction. Here we report a novel silk noil-based three-dimensional (3D) nonwoven scaffold produced by carding and needling with no formic acid exposure, which might improve burn healing. Once wetted, it displays human skin-like physical features and a high biocompatibility. Human keratinocyte-like cervical carcinoma C4-I cells seeded onto the carded-needled nonwovens in vitro quickly adhered to them, grew, and actively metabolized glutamine releasing lactate. As on plastic, they released no proinflammatory IL-1ß, although secreting tumor necrosis factor-alpha, an inducer of the autocrine mitogen amphiregulin in such cells. Once grafted into interscapular subcutaneous tissue of mice, carded-needled nonwovens guided the afresh assembly of a connective tissue enveloping the fibroin microfibers and filling the interposed voids within 3 months. Fibroblasts and a few poly- or mononucleated macrophages populated the engineered tissue. Besides, its extracellular matrix contained thin sparse collagen fibrils and a newly formed vascular network whose endothelin-1-expressing endothelial cells grew first on the fibroin microfibrils and later expanded into the intervening matrix. Remarkably, no infiltrates of inflammatory leukocytes and no packed collagen fibers bundles among fibroin microfibers, no fibrous capsules at the grafts periphery, and hence no foreign body response was obtained at the end of 3 months of observation. Therefore, we posit that silk noil-based 3D carded-needled nonwoven scaffolds are tools for translational medicine studies as they could guide connective tissue regeneration at deep burn wounds averting scar retraction with good functional results.

Calcium-Sensing Receptors of Human Neural Cells Play Crucial Roles in Alzheimer's Disease.

In aged subjects, late-onset Alzheimer's disease (LOAD) starts in the lateral entorhinal allocortex where a failure of clearance mechanisms triggers an accumulation of neurotoxic amyloid-ß42 oligomers (Aß42-os). In neurons and astrocytes, Aß42-os enhance the transcription of Aß precursor protein (APP) and ß-secretase/BACE1 genes. Thus, by acting together with γ-secretase, the surpluses of APP and BACE1 amplify the endogenous production of Aß42-os which pile up, damage mitochondria, and are oversecreted. At the plasmalemma, exogenous Aß42-os bind neurons' and astrocytes' calcium-sensing receptors (CaSRs) activating a set of intracellular signaling pathways which upkeep Aß42-os intracellular accumulation and oversecretion by hindering Aß42-os proteolysis. In addition, Aß42-os accumulating in the extracellular milieu spread and reach mounting numbers of adjacent and remoter teams of neurons and astrocytes which in turn are recruited, again via Aß42-os•CaSR-governed mechanisms, to produce and release additional Aß42-os amounts. This relentless self-sustaining mechanism drives AD progression toward upper cortical areas. Later on accumulating Aß42-os elicit the advent of hyperphosphorylated (p)-Tau oligomers which acting together with Aß42-os and other glial neurotoxins cooperatively destroy wider and wider cognition-related cortical areas. In parallel, Aß42-os•CaSR signals also elicit an excess production and secretion of nitric oxide and vascular endothelial growth factor-A from astrocytes, of Aß42-os and myelin basic protein from oligodendrocytes, and of proinflammatory cytokines, nitric oxide and (likely) Aß42-os from microglia. Activated astrocytes and microglia survive the toxic onslaught, whereas neurons and oligodendrocytes increasingly die. However, we have shown that highly selective allosteric CaSR antagonists (calcilytics), like NPS 2143 and NPS 89626, efficiently suppress all the neurotoxic effects Aß42-os•CaSR signaling drives in cultured cortical untransformed human neurons and astrocytes. In fact, calcilytics increase Aß42 proteolysis and discontinue the oversecretion of Aß42-os, nitric oxide, and vascular endothelial growth factor-A from both astrocytes and neurons. Seemingly, calcilytics would also benefit the other types of glial cells and cerebrovascular cells otherwise damaged by the effects of Aß42-os•CaSR signaling. Thus, given at amnestic minor cognitive impairment (aMCI) or initial symptomatic stages, calcilytics could prevent or terminate the propagation of LOAD neuropathology and preserve human neurons' viability and hence patients' cognitive abilities.