Ubaldo Armato

Silk fibroin/poly(carbonate)-urethane as a substrate for cell growth: in vitro interactions with human cells

Silk fibroin (SF)-based or -coated biomaterials are likely to be endowed with structural and surface properties that render them particularly apt for biomedical applications. In this work we investigated the behavior of four different strains of normal human adult fibroblasts that had been seeded onto membranes made up of poly(carbonate) urethane (PCU), the surfaces of which had or had not been homogeneously coated with SF. Cell adhesion within 3h to the SF-coated PCU films was 2.2-fold that to their uncoated homologues. After 30 days of incubation in vitro, 2.5-fold more cells had grown on the SF-coated specimens than on the uncoated ones. This enhanced cell adherence and hence growth on the SF-coated surfaces was coupled with higher cumulative rates of D-glucose (but not L-glutamine) uptake and of both lactate and interleukin-6 (IL-6) cumulative secretion. Conversely, human fibroblasts cultured on either type of PCU scaffolds never secreted any ELISA-assayable amount of three main proinflammatory cytokines, namely interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1). Finally, when the metabolic activities were compared on a per 10(5) cells basis, it became clear that the adhesion to SF favored an initially higher consumption of D-glucose, a late higher release of IL-6, and an at-first more intense, but declining, extracellular assembly of type I collagen fibers. Overall, these results show that SF-coated PCU membranes represent a novel type of biomaterial that favors the adhesion, the growth and performance of specific metabolic tasks by normal human adult fibroblasts without eliciting any concurrent secretion of some of the chief proinflammatory cytokines.

Silk Fibroin-Coated Three-Dimensional Polyurethane Scaffolds for Tissue Engineering: Interactions with Normal Human Fibroblasts

Silk fibroin (SF)-based or -coated biomaterials hold structural and surface properties that render them suitable for biomedical applications. In this work, we investigated the behavior of four strains of normal human adult fibroblasts (HAFs) seeded onto polyurethane foam, uncoated (PUF) or SF coated (PUF/SF). HAF adhesion within 3 h to PUF/SF was 2-fold that of adhesion to PUF. After 30 days of incubation in vitro, 37% more HAFs had grown on PUF/SF than on PUF. Taking 10(5) cells as a basis for comparisons, HAFs on PUF/SF exhibited initially higher glucose consumption rates, but persistently lower glutamine uptake rates than on PUF, whereas the rates of lactate and interleukin 6 release and of extracellular assembly of type I collagen fibers were alike on either substrate. Moreover, HAFs on both PUF/SF and PUF never secreted any ELISA-assayable amounts of interleukin 1beta, tumor necrosis factor alpha, and transforming growth factor beta(1). Hence, PUF/SF scaffolds embody a novel class of biomaterials favoring the adhesion, proliferation, and performance of specific metabolic tasks by HAFs without eliciting any concurrent secretion of the chief proinflammatory cytokines.

C-terminal fragment of parathyroid hormone-related protein, PTHrP-(107-111), stimulates membrane-associated protein kinase C activity and modulates the proliferation of human and murine skin keratinocytes.

Low concentrations of the C‐terminal parathyroid hormone‐related protein (PTHrP) fragments, PTHrP‐(107–111) and PTHrP‐(107–139), stimulated membrane‐associated protein kinase Cs (PKCs), but not adenylyl cyclase or an internal Ca2+ surge, in early passage human skin keratinocytes and BALB/MK‐2 murine skin keratinocytes. The fragment maximally stimulated membrane‐associated PKCs in BALB/MK‐2 cells at 5 × 10−9 to 10−8 M. The maximally PKC‐stimulating concentrations of PTHrP‐(107–111) also stopped or stimulated BALB/MK‐2 keratinocyte proliferation depending on whether the cells were, respectively, cycling or quiescent at the time of exposure. Thus, just one brief (30‐minute) pulse of 10 −8 M PTHrP‐(107–111) stopped the proliferation of BALB/MK‐2 keratinocytes for at least 5 days. On the other hand, daily 30‐minute pulses of 10−8 M PTHrP‐(107–111) started and then maintained the proliferation of initially quiescent BALB/MK‐2 cells. Similarly PTHrP‐(107–111) inhibited DNA synthesis by cycling primary adult human keratinocytes, but it stimulated DNA synthesis by quiescent human keratinocytes.

Roles of Ca2+ and the Ca2+‐sensing receptor (CASR) in the expression of inducible NOS (nitric oxide synthase)‐2 and its BH4 (tetrahydrobiopterin)‐dependent activation in cytokine‐stimulated adult human astrocytes

Since NO production by NOS‐2 made by astrocytes activated by proinflammatory cytokines contributes to the killing of neurons in variously damaged human brains, knowing the mechanisms responsible for NOS‐2 expression should contribute to developing effective therapeutics. The expression and activation of NOS‐2 in normal adult human cerebral cortical astrocytes treated with three proinflammatory cytokines, IL‐1β, TNF‐α, and IFN‐γ, are driven by two separable mechanisms. NOS‐2 expression requires a burst of p38 MAPK activity, while the activation of the resulting enzyme protein requires MEK/ERK‐dependent BH4 (tetrahydrobiopterin) synthesis between 24 and 24.5 h after adding the cytokines to the culture medium. Here we show that NOS‐2 expression in the activated astrocytes requires that the culture medium contain 1.8 mM Ca2+, but it is unaffected by inhibiting calcium‐sensing receptors (CASRs) with NPS 89636. However, NOS‐2 activation is inhibited by NPS 89626 during the MEK/ERK‐dependent stage between 24 and 24.5 h after adding the cytokines, and this inhibition can be overridden by exogenous BH4. Therefore, NOS‐2 expression and the subsequent BH4‐dependent NOS‐2‐activation in human astrocytes need 1.8 mM Ca2+ to be in the culture medium, while NOS‐2 activation also needs functional CASRs between 24 and 24.5 h after cytokine addition. These findings raise the possibility that calcilytic drugs prevent NO‐induced damage and death of human neurons.

Silk fibroin-polyurethane scaffolds for tissue engineering

Silk fibroin (SF) is a highly promising protein for its surface and structural properties, associated with a good bio- and hemo-compatibility. However, its mechanical properties and architecture cannot be easily tailored to meet the requirements of specific applications.In this work, SF was used to modify the surface properties of polyurethanes (PUs), thus obtaining 2D and 3D scaffolds for tissue regeneration. PUs were chosen for their well known advantageous properties and versatility; they can be obtained either as 2D (films) or 3D (foams) substrates. Films of a medical-grade poly-carbonate-urethane were prepared by solvent casting; PU foams were purposely designed and prepared with a morphology (porosity and cell size) adequate for cell growth. PU substrates were coated with fibroin by a dipping technique. To stabilize the coating layer, a conformational change of the protein from the α-form (water soluble) to the β-form (not water soluble) was induced.Novel methodology in UV spectroscopy were developed for quantitatively analyzing the SF-concentration in dilute solutions. Pure fibroin was used as standard, as an alternative to the commonly used albumin, allowing real concentration values to be obtained.SF-coatings showed good stability in physiological-like conditions. A treatment with methanol further stabilized the coating.Preliminary results with human fibroblasts indicated that SF coating promote cell adhesion and growth, suggesting that SF-modified PUs appear to be suitable scaffolds for tissue engineering applications.© 2001 Kluwer Academic Publishers

Hippocampal Membrane-Associated p75NTR Levels are Increased in Alzheimer's Disease

The pan-specific p75 neurotrophin receptor (p75(NTR)) is believed to play an important role in the pathogenesis of Alzheimers disease (AD). It is involved in mediating amyloid-β (Aβ) toxicity and stimulating amyloidogenesis. In addition, we have recently shown that stimulating cultured SH-SY5Y human neuroblastoma cells with Aβ(42) increases the level of membrane-associated p75(NTR) and that Aβ(42)-accumation in two strains of transgenic AD model mice is accompanied by an increased level of hippocampal membrane-associated p75(NTR) (Chakravarthy et al. J Alzheimers Dis 19, 915-925, 2010). This raised an important question whether accumulating Aβ(42) in human AD is also accompanied by an increased hippocampal membrane-associated p75(NTR). In this study, using polyclonal and monoclonal antibodies against the p75(NTR) receptors intra- and extracellular domains, we show that indeed the mean level of membrane-associated p75(NTR) in the hippocampal formation is significantly higher (~two-fold, p < 0.03) in human AD brains than in identical samples of hippocampal formation in age-matched non-AD human brains. The possible relation of this elevated hippocampal p75(NTR) to AD cognitive decline is discussed.

Nitric oxide in the liver: Physiopathological roles

Many of the known roles of arginine (e.g. in immune function, wound healing, and protection against ammonia intoxication) are mediated by a metabolic pathway synthesising nitric oxide (NO) in the liver. Contrary to some of the current views, liver-produced NO may be basically beneficial, as it exerts both protective actions against tissue injury and cytotoxic effects on invading microorganisms, parasites, or tumor cells. An ongoing equilibrium between NO and other NO-reactive compounds (e.g. O2 and non-heme iron-sulphur-containing moieties) appears to be important in this respect, even under critical conditions. Thus, NO may prevent liver tissue harm from oxidant stress. Only when this putative counterbalance is upset by an uncontrolled, prolonged and/or massive production of NO, liver tissue damage may occur leading to hepatic inflammation or even tumor development. Moreover, the currently available data support the working hypothesis that hepatocytes partake not only to immunoregulatory processes, but even to immune defence mechanisms. Thus, the liver constitutes an excellent model for investigations into the crosstalks regulating the production of NO which take place among not only the various networks operating inside a single hepatic cell, but even the individual types of liver cells.

Do Astrocytes Collaborate with Neurons in Spreading the “Infectious” Aβ and Tau Drivers of Alzheimer’s Disease?

Evidence has begun emerging for the “contagious” and destructive Aβ42 (amyloid-beta42) oligomers and phosphorylated Tau oligomers as drivers of sporadic Alzheimer’s disease (AD), which advances along a pathway starting from the brainstem or entorhinal cortex and leading to cognition-related upper cerebral cortex regions. Seemingly, Aβ42 oligomers trigger the events generating the neurotoxic Tau oligomers, which may even by themselves spread the characteristic AD neuropathology. It has been assumed that only neurons make and spread these toxic drivers, whereas their associated astrocytes are just janitorial bystanders/scavengers. But this view is likely to radically change since normal human astrocytes freshly isolated from adult cerebral cortex can be induced by exogenous Aβ25-35, an Aβ42 proxy, to make and secrete increased amounts of endogenous Aβ42. Thus, it would seem that the steady slow progression of AD neuropathology along specific cognition-relevant brain networks is driven by both Aβ42 and phosphorylated Tau oligomers that are variously released from increasing numbers of “contagion-stricken” members of tightly coupled neuron–astrocyte teams. Hence, we surmise that stopping the oversecretion and spread of the two kinds of “contagious” oligomers by such team members, perhaps via a specific CaSR (Ca2+-sensing receptor) antagonist like NPS 2143, might effectively treat AD.

The Aβ Peptides-Activated Calcium-Sensing Receptor Stimulates the Production and Secretion of Vascular Endothelial Growth Factor-A by Normoxic Adult Human Cortical Astrocytes

The excess vascular endothelial growth factor (VEGF) produced in the Alzheimer’s disease (AD) brain can harm neurons, blood vessels, and other components of the neurovascular units (NVUs). But could astrocytes partaking in networks of astrocyte-neuron teams and connected to blood vessels of NVUs contribute to VEGF production? We have shown with cultured cerebral cortical normal (i.e., untransformed) adult human astrocytes (NAHAs) that exogenous amyloid-β peptides (Aβs) stimulate the astrocytes to make and secrete large amounts of Aβs and nitric oxide by a mechanism mediated through the calcium-sensing receptor (CaSR). Here, we report that exogenous Aβs stimulate the NAHAs to produce and secrete even VEGF-A through a CaSR-mediated mechanism. This is indicated by the ability of Aβs to specifically bind the CaSR, and the capability of a CaSR activator, the “calcimimetic” NPS R-568, to imitate, and of the CaSR antagonist, “calcilytic” NPS 2143, to inhibit, the Aβs stimulation of VEGF-A production and secretion by the NAHAs. Thus, Aβs that accumulate in the AD brain may make the astrocytes that envelop and functionally collaborate with neurons into multi-agent AD-driving “machines” via a CaSR signaling mechanism(s). These observations suggest the possibility that CaSR allosteric antagonists such as NPS 2143 might impede AD progression.

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action

Trimers of bovine pancreatic RNase A were obtained by cross‐linking native RNase A with dimethyl suberimidate. They degrade double‐stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4‐I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross‐linked dimers of RNase A appear to be ineffective.