New vandetanib analogs: fused tricyclic quinazolines with antiangiogenic potential.

The antiangiogenic effects of three novel anilinoquinazoline derivatives were studied with the aim to find new multi-kinase inhibitors as anticancer agents. The compounds are characterized by dioxolane, dioxane and dioxepine rings and bear the same aniline substituent in 4 position as vandetanib, known antiangiogenic agent. The in vitro assays were carried out on human umbilical vein endothelial cells (HUVECs), whereas in vivo angiogenesis was evaluated by means of Matrigel plug assay. The results showed that these compounds exert, even though to different extents, antiangiogenic activity affecting the various step of the process that leads to the formation of new blood vessels. At high concentrations they induced antiproliferative effects, whereas at non-cytotoxic concentrations they inhibited cell migration and the formation of tubular structures in Matrigel. In in vitro assays the dioxolane derivative 1 was more effective than vandetanib. Indeed, it inhibited the effects induced by exogenous VEGF and FGF-2 on both cell proliferation and morphogenesis, whereas vandetanib was completely ineffective. Moreover, all the compounds, as vandetanib, counteracted the FGF-2-induced increase in the hemoglobin content in the Matrigel plugs. Our results showed that all the three novel derivatives possess both in vitro and in vivo antiangiogenic activity, with compound 1 more effective than vandetanib to inhibit in vitro angiogenesis induced by exogenous cytokines.

Porous alginate/poly(ε-caprolactone) scaffolds: preparation, characterization and in vitro biological activity.

In bone tissue engineering, scaffolds with controlled porosity are required to allow cell ingrowth, nutrient diffusion and sufficient formation of vascular networks. The physical properties of synthetic scaffolds are known to be dependent on the biomaterial type and its processing technique. In this study, we demonstrate that the separation phase technique is a useful method to process poly(ε-caprolactone) (PCL) into a desired shape and size. Moreover, using poly(ethylene glycol), sucrose, fructose and Ca2+ alginate as porogen agents, we obtained PCL scaffolds with three-dimensional porous structures characterized by different pore size and geometry. Scanning electron microscopy and porosity analysis indicated that PCL scaffolds prepared with Ca2+ alginate threads resemble the porosity and the homogeneous pore size distribution of native bone. In parallel, MicroCT analysis confirmed the presence of interconnected void spaces suitable to guarantee a biological environment for cellular growth, as demonstrated by a biocompatibility test with MC3T3-E1 murine preosteoblastic cells. In particular, scaffolds prepared with Ca2+ alginate threads increased adhesion and proliferation of MC3T3-E1 cells under basal culture conditions, and upon stimulation with a specific differentiation culture medium they enhanced the early and later differentiated cell functions, including alkaline phosphatase activity and mineralized extracellular matrix production. These results suggest that PCL scaffolds, obtained by separation phase technique and prepared with alginate threads, could be considered as candidates for bone tissue engineering applications, possessing the required physical and biological properties.

Effects of beta-amyloid on rat neuromicrovascular endothelial cells cultured in vitro.

Several studies have shown that beta-amyloid (beta A) deposits are associated with damage of cerebral vessels and that in Alzheimer's disease (AD) beta A peptides are cytotoxic for cerebral endothelial cells (ECs). However, little is known about the mechanisms underlying these effects of beta A peptides. Hence, we have investigated the effects of beta A(1-40) and beta A(1-42) on rat neuromicrovascular ECs (NECs) cultured in vitro. NECs were isolated, plated (1.5x10(4) cells/cm2) on collagen/fibronectin-coated Petri dishes and cultured in EC growth medium MV2. After 24 h of culture, NECs were incubated with beta A(1-40) and beta A(1-42) (10(-9) or 10(-7) M) and cultured for another 3, 24 or 48 h. Cell viability was assayed by either trypan blue exclusion or by measuring redox activity (MTS assay). Cell proliferation was assessed by measuring the incorporation of 5'-bromo-2'-deoxyuridine into DNA, cell apoptosis by TUNEL assay and cell necrosis by evaluating the release of lactate dehydrogenase. The morphology of cultured NECs was examined by transmission electron microscopy. Other NECs were plated (2.5x10(4) cells/cm2) on Matrigel coated-wells and incubated for 18 h in the presence or absence of beta A peptides for in vitro angiogenesis assay. Beta A peptides significantly decreased NEC viability and enhanced cell apoptosis and necrosis rates. NEC proliferation was not significantly affected. The effects were seen after an incubation period of 3 h (and also 24 h in the case of the redox activity) but not 48 h and beta A(1-42) was much more effective in its toxic effects than beta A(1-40). NECs incubated for 24 h with beta A peptides displayed ultrastructural signs of cell degeneration. beta A peptides prevented NECs cultured on Matrigel to form a capillary-like network and image analysis showed that the downloading of dimensional and topological parameters of the meshwork was significant only in the case of the incubation with beta A(1-42). Collectively our findings allow us to conclude that i) beta A peptides exert a marked toxic effect on cultured NECs, which conceivably reduces their in vitro angiogenic activity; ii) beta A(1-42) is the more toxic form, which could suggest its main role in the pathogenesis of cerebral vessel lesions in AD and iii) the maximum toxic action occurs after a short incubation period, which could be explained by assuming that beta A peptides are unable to accumulate in NECs due to their rapid degradation, thereby allowing NECs to fully recover within 48 h.

Angiogenic response induced by acellular aortic matrix in vivo.

In this study, we investigated the angiogenic response induced by acellular aortic matrices implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. Results showed that acellular matrices were able to induce a strong angiogenic response comparable to that of fibroblast growth factor 2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta 1 (TGF-beta1) were added to the matrices and inhibited by the addition of an anti-FGF-2 or anti-TGF-beta1 antibodies. The response may be considered dependent on a direct angiogenic effect exerted by the matrices and in part also by the presence of FGF-2 and TGF-beta1 in the acellular matrices.

Biological fate of tissue-engineered porcine valvular conduits xenotransplanted in the sheep thoracic aorta.

The ideal prosthesis to replace the diseased human aortic valve is not yet available. We have previously shown that porcine acellular aortic-valve conduits, obtained by detergent-enzymatic method, display hemodynamic performances similar to those of their native counterparts. Hence, it seemed worthwhile to ascertain whether these tissue-engineered prostheses can be successfully xenotransplanted. Porcine acellular conduits, which immunocytochemistry demonstrated to lack MHC class I and II antigens, were implanted in the thoracic aorta of 9 sheep. Two animals died just after surgery, and the other 7 sheep were sacrificed 1 or 5 months after transplantation. A rather favorable outcome of the implant was observed in 4 sheep. In these animals, aortic valves remained pliable and coaptive, and the luminal surface of the conduits was endothelized just after one month from surgery. An intense inflammatory response was present at 1 month, and, although attennuated, it persisted for 5 months, located mainly between the tunica intima and media and at the border of the implant. Vimentin-positive and smooth muscle actin-positive myofibroblasts proliferated within tunica media and adventitia, and an obvious thickening of the tunica intima was also observed. Small vessels were seen in the adventitia, and elastic fibers were well-preserved in both the aorta wall and valve leaflets. In the cases of unfavorable outcome (3 of 7 survived sheep), implants were detached from the aorta recipient and surrounded by a connective mass that almost completely obstructed their lumen. These masses were composed of a fibromyxoid background where proliferating cells, resembling those occurring in human reactive myofibroblastic lesions (proliferative fascitis), were embedded. Collectively, these rather disappointing findings indicate that acellular valve conduits, obtained by the detergent-enzymatic method, are presently not suitable for clinical applications because of the persistent inflammatory response, which conceivably triggers overgrowth mechanisms that lead to implant failure.

Adrenomedullin and endothelin-1 stimulate in vitro expansion of cord blood hematopoietic stem cells.

The improvement of techniques for in vitro expansion of cord blood (CB) hematopoietic stem cells (SCs) is, at present, one main task of tissue engineering. Hence, we investigated whether endothelin-1 (ET-1) and adrenomedullin (AM), two regulatory peptides exerting growth promoting action on several cell systems, favor the in vitro expansion of CB SCs in liquid culture. CB hematopoietic cell middle-term expansion was carried out in a stroma-free liquid culture medium in the presence of ET-1, AM and three different cytokine combinations. After two weeks of incubation, aliquots of expanded-cell suspension were seeded on semisolid medium and clonogenic tests were carried out by counting the number of colony forming units (CFUs) after 14 days of culture. Neither ET-1 nor AM (2.5 x 10(-8) M) were per se able to significantly increase the CFU number, but both peptides magnified the pro-expansive effects of some cytokine cocktails. In light of these findings, we conclude that ET-1 and AM are to be considered novel promising molecules that, in association with cytokines, can be utilized as pro-expansive factors of CB SCs in prevision of their clinical use in allogeneic transplantation.

Effects of prostaglandins E1 and E2 on the growth and differentiation of osteoblast-like cells cultured in vitro.

Prostaglandins E (PGEs) are abundantly produced in the skeletal tissue, the turnover of which they can modulate acting on both bone deposition and resorption. We compared the effects of PGE1 and PGE2 on the growth and differentiation of rat bone-marrow osteoblast-like cells cultured in vitro. Both PGEs stimulated cultured cell growth, PGE2 being more effective than PGE1. PGE1 inhibited and PGE2 enhanced alkaline phosphatase activity. Both PGEs markedly raised osteocalcin synthesis, without apparently affecting collagenase-digestible protein production. Scanning electron microscopy showed that untreated cultured osteoblast-like cells were arranged in clusters and displayed a polygonal shape. PGE1 did not alter cell morphology, while PGE2 provoked elongation of cultured cells and sprouting of slend cytoplasmic processes. Morphometric analysis indicated that PGE1 decreased and PGE2 increased cultured-cell dimensions. Collectively, these findings allow us to conclude that PGE1 and PGE2, although being both able to enhance proliferation of osteoblast-like cells cultured in vitro, exert divergent effects on their differentiation. PGE1 seems to slow-down osteoblast maturation, while PGE2 appears to stimulate osteoblast differentiation to mature osteocytes.