3D bioengineered neural tissue generated from patient-derived iPSCs mimics time-dependent phenotypes and transcriptional features of Alzheimer's disease.

Several iPSC-derived three-dimensional (3D) cultures have been generated to model Alzheimer's disease (AD). While some AD-related phenotypes have been identified across these cultures, none of them could recapitulate multiple AD-related hallmarks in one model. To date, the transcriptomic features of these 3D models have not been compared with those of human AD brains. However, these data are crucial to understanding the pertinency of these models for studying AD-related pathomechanisms over time. We developed a 3D bioengineered model of iPSC-derived neural tissue that combines a porous scaffold composed of silk fibroin protein with an intercalated collagen hydrogel to support the growth of neurons and glial cells into complex and functional networks for an extended time, a fundamental requisite for aging studies. Cultures were generated from iPSC lines obtained from two subjects carrying the familial AD (FAD) APP London mutation, two well-studied control lines, and an isogenic control. Cultures were analyzed at 2 and 4.5 months. At both time points, an elevated Aß42/40 ratio was detected in conditioned media from FAD cultures. However, extracellular Aß42 deposition and enhanced neuronal excitability were observed in FAD culture only at 4.5 months, suggesting that extracellular Aß deposition may trigger enhanced network activity. Remarkably, neuronal hyperexcitability has been described in AD patients early in the disease. Transcriptomic analysis revealed the deregulation of multiple gene sets in FAD samples. Such alterations were strikingly similar to those observed in human AD brains. These data provide evidence that our patient-derived FAD model develops time-dependent AD-related phenotypes and establishes a temporal relation among them. Furthermore, FAD iPSC-derived cultures recapitulate transcriptomic features of AD patients. Thus, our bioengineered neural tissue represents a unique tool to model AD in vitro over time.

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells.

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Chronic Resveratrol Treatment Reduces the Pro-angiogenic Effect of Human Fibroblast "Senescent-Associated Secretory Phenotype" on Endothelial Colony-Forming Cells: The Role of IL8.

Senescent cells are characterized by an increased secretion of inflammatory and growth factors, known as the "senescence-associated secretory phenotype" (SASP), producing a pro-tumoral and pro-angiogenic microenvironment. This work proposes chronic resveratrol treatment (5 µM for 5 weeks, termed R5) of senescent MRC5 fibroblasts as a mean to mimic and target the angiogenic trait of stromal fibroblast SASP. Senescent fibroblast conditioned medium (CM sen) was effective in enhancing the angiogenic properties of endothelial colony-forming cells (ECFCs), that is, invasive activity and capillary morphogenesis capability in vitro, that were significantly reduced when conditioned media were collected after resveratrol pretreatment (CM senR5). The attenuation of ECFC angiogenic phenotype induced by CM senR5 was accompanied by reduced protein levels of epidermal growth factor and urokinase plasminogen activator receptors (EGFR, uPAR), and by a related decreased activation of receptor-tyrosine-kinase signaling pathways. IL8 levels were found reduced in CM senR5 compared to CM sen, with the associated reduction of IL8-CXCR2 binding in ECFCs. IL8-subtraction mitigated the pro-angiogenic features of CM sen and the associated intracellular signaling in ECFCs, indicating a prominent role of IL8 in the pro-angiogenic effects of CM sen. IL8 modulation is an important mechanism underlying the antiangiogenic activity of resveratrol on MRC5 SASP.

Serpin A1 and the modulation of type I collagen turnover: Effect of the C-terminal peptide 409-418 (SA1-III) in human dermal fibroblasts.

The pharmacological modulation of collagen turnover is a strategy potentially useful in different skin conditions. The serine protease inhibitor Serpin A1 and portions of its C-terminal region have been investigated as collagen modulators. To clarify the mechanisms by which the C-terminal 409-418 peptide SA1-III increases extracellular type I collagen levels, to compare its activities range with that of the originator molecule Serpin A1, and to evaluate its efficacy in primary cultures from adult and aged human subjects. The different forms of type I collagen were analyzed by means of western blot in cell lysates and cell-conditioned media of primary human dermal fibroblasts obtained from subjects of different ages. Gelatin zymography was used to investigate the degrading enzymes. Cell viability and in vitro wound healing tests were used to evaluate cell proliferation. The SA1-III peptide increased extracellular collagen levels by reducing degradation, with no effect on cellular biosynthesis or cell proliferation mechanisms. A reduced level of MMP-2 and MMP-9 was also found in cell media upon peptide treatment. No peptide effect was detected on inflammatory mediators gene expression in resting and LPS-stimulated fibroblasts, or in the wound healing test. The SA1-III peptide is a good collagen modulator candidate, protecting collagen against degradation without detectable actions on biosynthesis, acting at reasonably low concentrations, and non-interfering with cell proliferation. It is effective in primary fibroblasts from young and aged subjects. These effects can prove useful in pathological and physiological skin conditions in which collagen degradation is excessive compared to the synthetic capacity.

Modulation of the Senescence-Associated Inflammatory Phenotype in Human Fibroblasts by Olive Phenols.

Senescent cells display an increase in the secretion of growth factors, inflammatory cytokines and proteolytic enzymes, termed the "senescence-associated-secretory-phenotype" (SASP), playing a major role in many age-related diseases. The phenolic compounds present in extra-virgin olive oil are inhibitors of oxidative damage and have been reported to play a protective role in inflammation-related diseases. Particularly, hydroxytyrosol and oleuropein are the most abundant and more extensively studied. Pre-senescent human lung (MRC5) and neonatal human dermal (NHDF) fibroblasts were used as cellular model to evaluate the effect of chronic (4-6 weeks) treatment with 1 µM hydroxytyrosol (HT) or 10 µM oleuropein aglycone (OLE) on senescence/inflammation markers. Both phenols were effective in reducing ß-galactosidase-positive cell number and p16 protein expression. In addition, senescence/inflammation markers such as IL-6 and metalloprotease secretion, and Ciclooxigenase type 2 (COX-2) and α-smooth-actin levels were reduced by phenol treatments. In NHDF, COX-2 expression, Nuclear Factor κ-light-chain-enhancer of activated B cells (NFκB) protein level and nuclear localization were augmented with culture senescence and decreased by OLE and HT treatment. Furthermore, the inflammatory effect of Tumor Necrosis Factor α (TNFα) exposure was almost completely abolished in OLE- and HT-pre-treated NHDF. Thus, the modulation of the senescence-associated inflammatory phenotype might be an important mechanism underlying the beneficial effects of olive oil phenols.

Chronic Resveratrol Treatment Inhibits MRC5 Fibroblast SASP-Related Protumoral Effects on Melanoma Cells.

Cellular senescence is related to organismal aging and is observed after DNA damaging cancer therapies, that induce tumor-suppressive modifications, but it is characterized by a strong increase in secreted factors, termed the "senescence-associated secretory phenotype" (SASP). Particularly, SASP from stroma senescent fibroblasts creates a cancer-favoring microenvironment, providing targets for anti-cancer interventions. In the present article, chronic treatment (5 weeks) with 5 µM resveratrol has been used to modulate senescence-related protumoral features of MRC5 fibroblasts, reducing SASP-related interleukins IL1α, IL1ß, IL6, and IL8; transforming-growth-factor-ß (TGFß); matrix metallo-proteinases MMP3 and MMP2; urokinase plasminogen activator (uPA); receptor proteins uPAR, IL6R, insulin growth factor receptor-1 (IGF-1R), TGFß-R2, and CXCR4. The cellular nuclear-factor-kB (NF-kB) protein level was also reduced, confirming its role in the induction of SASP. Resveratrol pretreated MRC5 fibroblasts were resistant to activation by TGFß. Resveratrol treatment of senescent MRC5 induced the production of conditioned media (CM) which counteracted the protumoral effect of senescent CM on A375 and A375-M6 melanoma cell proliferation and invasiveness, and reduced the expression of epithelial-to-mesenchymal transition markers related to malignant features. This experimental approach proposes a treatment that targets the senescent stromal cell phenotype to induce an anti-tumor hosting microenvironment, which is suitable for both preventive and therapeutic purposes.