Tissue engineering can effectively contribute to the development of novel vascular prostheses aimed to overcome the well-known drawbacks of small-diameter grafts. To date, poly(ε-caprolactone) (PCL), a bioresorbable synthetic poly(α-hydroxyester), is considered one of the most promising materials for vascular tissue engineering. In this work, the potential advantage of intimate blending soft PCL and hard poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a polymer of microbial origin, has been evaluated. Nonwoven mats and small-diameter tubular scaffolds of PCL, PHBV, and PCL/PHBV were fabricated by means of electrospinning technique. Mechanical properties and suture retention strength were investigated according to the international standard for cardiovascular implants. Biological tests demonstrated that both PCL-based scaffolds supported survival and growth of rat cerebral endothelial cells in a short time. The fiber alignment of the electrospun tubular scaffolds contributed to a more rapid and homogeneous cell colonization of the luminal surface.

This work focuses on the evaluation of the potential use of electrospun poly(epsilon-caprolactone) (PCL) micrometric and/or sub-micrometric fibrous membranes for rat hippocampal astrocyte (HA) and rat cerebro-microvascular endothelial cell (CEC) cultures. Both mats supported cell adhesion, proliferation, cellular phenotype and spreading. Microfibrous mats allowed cellular infiltration, while both HAs and CECs were unable to migrate within the sub-micrometric fibrous mat, leaving an acellularized inner region. This finding was correlated to the presence of larger voids within electrospun PCL microfibrous mats, suggesting that the morphology should be accurately selected for the realization of a cell environment-mimicking mat. Based on our results, the proper fiber architecture can be regarded as a crucial issue to be considered in order to deal with suitable polymeric mats tailored for specific in vitro application.

Although Alzheimer's disease (AD) is considered a neurodengenerative disorders, in the last few years a large amount of evidence has suggested that it is also a vascular pathology characterized by increased capillary density and expression of angiogenic factors. In AD the endothelium degenerates, promoting local neuroinflammation and activation of brain endothelium, perivascular microglia, pericytes, astrocytes. Excess tumor necrosis factor (TNF) in the cerebrospinal fluid (CSF), at a concentration of 25 times higher than in the control group, has been demonstrated in AD. Recent studies provide evidence that treatment with TNF-α antagonists may result in a rapid cognitive improvement in AD patients. In the present work we investigated the role of astrocytes in AD angiogenesis and neuroinflammation by means of conditioned media of untreated and Aβ-treated rat hippocampal astrocytes (RHAs) on rat microvascular endothelial cells (RCECs). The results demonstrated that RHA media increase RCEC proliferation and capillary-like structure formation. Moreover RHAs secrete IL-1β and, only after the Aβ1-42 treatment, TNF-α promotes RCEC release of IL-1β, IL-6 and TNF-α. The removal of IL-1β, TNF-α and/or VEGF, a strong angiogenic inducer highly over-expressed in AD brains, by means of specific antibody-coated beads in RHA media affects RCEC release of IL-1β, IL-6 and TNF-α. We hypothesised that astrocytes contribute to AD angiogenesis and neuroinflammation by the direct release of pro-inflammatory cytokines. The effect of an anti-inflammatory agent, such as etanercept, decreased RCEC in vitro cytokine release. This could be compared to the effect found in our experiments with antibody anti TNF-α-coated beads.

Several studies have demonstrated that cerebrovascular dysfunction and damage play a significant role in the pathogenesis of Alzheimer disease (AD). In fact, ß-amyloid peptides (Aßs), the major component of the senile plaques and cerebrovascular amyloid deposits in AD, were shown to be cytotoxic to endothelial cells. We have recently observed that Aßs exert a toxic effect on neuromicrovascular endothelial cells (NECs) in a time-and concentration-dependent manner, apoptosis playing a pivotal role in this process. Hence, it seemed worthwhile to investigate the Aß-mediated apoptosis mechanism in NECs. Aßs were found to induce, after a short incubation period, apoptosis throughout caspase-8 activation. Moreover, Aßs elicited a highly significant (p<0.001) increase in superoxide dismutase (SOD) levels after a 3-h exposure period, while SOD concentration was not affected after a 24-h incubation. The time-dependent increase in SOD concentration is probably correlated with the production of an excess of reactive oxygen species. Collectively, our findings allow us to conclude that: i) Aßs may induce apoptosis via the activation of caspase-8, presumably by cross-linking and activating receptors of the death-receptor family; ii) oxidative stress is possibly involved in the Aß-induced cytotoxic effect; and iii) these two mechanisms do not act sequentially but, probably, are independent of each other.

Abstract. To ascertain whether the potential biological effects of ß amyloid (ßA) on the endothelium are partly mediated by the receptor for advanced glycation-end products (RAGE), we performed a series of experiments which analyzed the effects of the ßA peptide on in vitro cerebromicrovascular endothelial cells (CECs). Our results suggest that RAGE is directly responsible for ßA actions on CECs, such as its toxic effect on cell survival, viability and angiogenic capability. We observed that a 6-h incubation period exposing CECs to ßA increased the extracellular levels of nitrite. Furthermore, the presence of a nitric oxide synthase inhibitor, L-NAME, was able to enhance CEC survival and viability. Immunocytochemical analyses demonstrated that the peptide induced expression of the inducible form of NOS, iNOS, typically synthesized in response to immune/inflammatory stimuli. Upon blocking the interaction of ßA and RAGE, we observed significantly decreased levels of NO and suppression of iNOS immunoreactivity. In conclusion, our data suggest the involvement of RAGE, at least partly, in mediating the effects of ßA on CECs. In particular, the decrease of in vitro cell viability and functionality and nitrosative stress activation was inhibited by blocking ßA (1-42) -RAGE interaction.