Formation Of Vessel-like Structures Research Articles

Differentiation of C57/BL6 mice bone marrow mononuclear cells into early endothelial progenitors cells in different culture conditions.

Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in vitro tube formation in tridimensional matrices. These cells are able to differentiate into mature endothelial cells and participate in the re-endothelization of damaged vessels. In this work, we tested different cultured media that can promote the proliferation and differentiation of mononuclear cells (MNCs) into early EPCs, with defined concentrations of growth factors and serum in order to establish a composition that may ensure us the reproducibility of our cultures. MNCs from mice bone marrow were cultivated using selective culture media containing DMEM or M199 supplemented with 10% FBS, VEGF, bFGF, and IGF, for 3, 7, and 14 days. Differentiation into early EPCs was analyzed using immunohistochemistry, FACS and western blotting and by functional parameters as uptake of ac-LDL, and formation of vessel-like structures. The cells cultivated with medium DMEM-M1 (DMEM plus VEGF, bFGF and IGF) expressed CD34, CD133, CD31, VEGFR2, and VE-Cadherin at all culture time-points with increased expression of these markers after 7 days. Only EPCs cultured for 30 days were able to form vessel-like structure. The uptake of ac-LDL was observed after 3, 7, 14, and 30 days, confirming the differentiation of mononuclear cells into early EPCs. DMEM-M1 was able to sustain MNCs proliferation and differentiation, increasing the expression of the characteristic EPC markers, allowing the expansion of early EPCs in culture in a similar way to that observed in commercial available media.

Coculture of stem cells from apical papilla and human umbilical vein endothelial cell under hypoxia increases the formation of three-dimensional vessel-like structures in vitro.

The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) whether the host vasculature can be induced to penetrate the bioengineered pulp replacement and conjoin. Major inductive molecules that participate in laying down blood vessels include vascular endothelial growth factor (VEGF), ephrinB2, and hypoxia-inducible factor 1α (HIF-1α). Being able to modulate the genes encoding these angiogenic molecules is a therapeutic target in pulp regeneration for endogenous blood vessel formation, prevention of graft rejection, and exclusion of infection. Once implanted inside the root canal, bioengineered pulp is subjected to severe hypoxia that causes tissue degeneration. However, short-term hypoxia is known to stimulate angiogenesis. Thus, it may be feasible to prime dental cells for angiogenic activity before implantation. Stem cells from apical papilla (SCAP) are arguably one of the most potent and versatile dental stem cell populations for bioengineering pulp in vitro. Our study aimed to investigate whether coculture of SCAP and human umbilical vein endothelial cells (HUVECs) under hypoxia promotes the formation of endothelial tubules and a blood vessel network. In addition, we clarified the interplay between the genes that orchestrate these important angiogenic molecules in SCAP under hypoxic conditions. We found that SCAP cocultured with HUVEC at a 1:5 ratio increased the number of endothelial tubules, tubule lengths, and branching points. Fluorescence staining showed that HUVEC formed the trunk of tubular structures, whereas SCAP located adjacent to the endothelial cell line, resembling the pericyte location. When we used CoCl2 (0.5 mM) to induce hypoxic environment, the expression of proteins, HIF-1α and VEGF, and transcript of ephrinB2 in SCAP was upregulated. However, minimal VEGF levels in supernatants of HUVEC and coculture Petri dishes were detected, suggesting that VEGF secreted by SCAP might be used by HUVEC to accelerate the formation of vessel-like structures. Taken together, we revealed that artificial hypoxia stimulates angiogenic responses in SCAP for possible use in engineering dental pulp replacements. Our results may help to delineate the optimal therapeutic target to promote angiogenesis so that future bioengineered pulp replacements integrate faster and permanently within the host.

Effects of Platelet-rich Plasma and Cell Coculture on Angiogenesis in Human Dental Pulp Stem Cells and Endothelial Progenitor Cells

IntroductionPlatelet-rich plasma (PRP) has been described as platelet concentrate. Growth factors released by activated platelets can improve wound vasculogenesis and enhance wound healing. In this study, we used PRP instead of serum to culture human dental pulp stem cells (hDPSCs) and endothelial progenitor cells (EPCs) and investigated revascularization ability. The effect of hDPSC and EPC coculture on vasculogenesis was also studied. MethodsPRP was prepared by secondary centrifugation. Real-time polymerase chain reaction and Western blotting were used to determine the expression of vasculogenesis-related factors vascular endothelial growth factor, platelet-derived growth factor, fetal liver kinase 1 (Flk-1), and stromal cell-derived factor 1 (SDF-1) in cultured hDPSCs and EPCs. The cells were divided into 4 groups: EPCs + 10% fetal bovine serum (FBS), EPCs + 10% PRP, EPCs + hDPSCs + 10% FBS, and EPCs + hDPSCs + 10% PRP. Then, the formation of vessel-like structures was tested by the tube formation assay. ResultsOn day 3, the expression levels of all the markers in the coculture groups were much higher than in the single-culture groups and were also higher in the PRP groups compared with the FBS groups (P < .05), except for SDF-1. Expression levels were significantly higher in the experimental groups (EPCs + 10% PRP, EPCs + hDPSCs + 10% FBS, and EPCs + hDPSCs + 10% PRP) than in the control group (EPCs + 10% FBS) and in the PRP groups/coculture groups compared with the FBS groups/single-culture groups (P < .01). The tube formation assay showed the area of vessel-like structures formed by the PRP group to be larger than in the FBS group (P < .05). ConclusionsPRP and coculture can both promote vasculogenesis, and PRP can promote EPCs to form vessel-like structures.

Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential.

BackgroundSeveral studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process.ResultsHerein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity.ConclusionsOur findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

In vitro and in vivo angiogenic capacity of BM-MSCs/HUVECs and AT-MSCs/HUVECs cocultures

The aim of this study was to comparatively evaluate the angiogenic capacity of cocultures using either human bone marrow- or human adipose tissue-derived mesenchymal stem cells (MSCs) (BM- or AT-MSCs) with human umbilical vein endothelial cells (HUVECs) both in vitro and in vivo at early time points (i.e. days 3 and 7). In vitro, cells were either monocultured (i.e. BM-MSCs, AT-MSCs or HUVECs) or cocultured (i.e. BM-MSCs/HUVECs and AT-MSCs/HUVECs) on Thermanox® (2-dimensional, 2D) or in collagen gels (3-dimensional, 3D). For the in vivo experiment, cells (cocultures) were embedded in collagen gels and implanted subcutaneously in nude mice. For both in vitro and in vivo experiments, samples were collected on days 3 and 7 and histologically processed for hematoxylin-eosin and platelet endothelial cell adhesion molecule (PECAM-1; CD31) staining. For in vivo samples, quantitative parameters for evaluating angiogenesis included CD31-positive staining percentage, total vessel-like structure (VLS) area percentage, VLS density, and average VLS area (i.e. the size of per VLS). In vitro results showed the formation of VLS in both cocultures, while none of the monocultures showed VLS formation, irrespective of 2D or 3D culture condition. Although VLS formation occurred after in vivo implantation, no significant difference in angiogenic capacity was observed between the two cocultures, either on day 3 or on day 7. Further, VLS density decreased and anastomosis of the new human vessels with the murine host vasculature occurred over time. In conclusion, this study demonstrated that AT-MSCs/HUVECs and BM-MSCs/HUVECs have equal angiogenic capacity both in vitro and in vivo, and that vessels from donor origin can anastomose with the host vasculature within seven days of implantation.

Endoglin is necessary for angiogenesis in human ovarian carcinoma-derived primary endothelial cells

Endoglin (CD105, END) is upregulated in proliferating endothelial cells, suggesting potential therapeutic properties. However, it is not clear whether endoglin mediates an enhanced proliferative rate or may be upregulated as part of a negative feedback loop. To gain insights into context-dependent and cell type-dependent regulatory effects of endoglin, we studied its role properties in human ovarian carcinoma-derived endothelial cells (ODMECs). We isolated and cultured primary ODMECs from epithelial ovarian carcinoma tissue. ODMECs had higher expression of endoglin and VEGFR-2, and also exhibited enhanced spontaneous formation of vessel-like structures in vitro. Transfection of siRNA targeting endoglin in ODMECs cells resulted in the reduction of the proliferation and tube formation. These results indicate that a subset of ODMECs display abnormal angiogenic properties and this phenotype was blocked by decreasing endoglin levels, suggesting endoglin is essential for stimulating angiogenesis, and targeting it may be an attractive approach to anti-angiogenesis therapy for ovarian carcinoma.

Paracrine exchanges of molecular signals between alginate-encapsulated pericytes and freely suspended endothelial cells within a 3D protein gel

Paracrine signals, essential for the proper survival and functioning of tissues, may be mimicked by delivery of therapeutic proteins within engineered tissue constructs. Conventional delivery methods are of limited duration and are unresponsive to the local environment. We developed a system for sustained and regulated delivery of paracrine signals by encapsulating living cells of one type in alginate beads and co-suspending these cell-loaded particles along with unencapsulated cells of a second type within a 3D protein gel. This system was applied to vascular tissue engineering by placing human placental microvascular pericytes (PCs) in the particulate alginate phase and human umbilical vein endothelial cells (HUVECs) in the protein gel phase. Particle characteristics were optimized to keep the encapsulated PCs viable for at least two weeks. Encapsulated PCs were bioactive in vitro, secreting hepatocyte growth factor, an angiogenic protein, and responding to externally applied HUVEC-derived signals. Encapsulated PCs influenced HUVEC behavior in the surrounding gel by enhancing the formation of vessel-like structures when compared to empty alginate bead controls. In vivo, encapsulated PCs modulated the process of vascular self-assembly by HUVECs in 3D gels following implantation into immunodeficient mice. We conclude that alginate encapsulated cells can provide functional paracrine signals within engineered tissues.

Deletion of integrin linked kinase in endothelial cells results in defective RTK signaling caused by caveolin 1 mislocalization

Integrin linked kinase (ILK) connects the ILK-Pinch-Parvin complex with integrin adhesion sites. Because of the functional relevance of integrin-linked signaling for endothelial cell (EC) biology, we have explored this pathway in Ilk(-/-) embryonic stem (ES) cells differentiated into ECs and vessel-like structures. We have focused in particular on the mechanistic relevance of ILK-Pinch-Parvin complex-related signaling for EC development and tube formation. Our analysis revealed that the formation of vessel-like structures was strongly reduced in Ilk(-/-) ES cells and that this phenotype could be rescued by re-expression of ILK in ES cells. ECs were MACS sorted from wild-type (WT) and Ilk(-/-) ES cells and functional analysis using intracellular calcium imaging as the read-out yielded a complete lack of vascular endothelial growth factor- and epidermal growth factor-dependent responses. The possibility of a caveolin 1-related defect was investigated by transfecting WT and Ilk(-/-) ECs with a caveolin 1-EGFP fusion protein. Time-lapse microscopy showed that the prominent phenotype is due to altered dynamics of caveolin 1 and to a lack of positioning of caveolin 1 in the vicinity of the plasma membrane and that it is rescued by re-expressing ILK in the Ilk(-/-) ES cells. We also found that the defect is caused by the perturbed organization of microtubules and cortical actin filaments. Thus, ILK is required as a scaffold to allow actin-microtubule interactions and correct positioning of caveolin 1 close to the plasma membrane. This is crucial for signaling compartmentalization in ECs and explains the key role of ILK for EC development and function.

Deletion of integrin linked kinase in endothelial cells results in defective RTK signaling caused by caveolin 1 mislocalization

Integrin linked kinase (ILK) connects the ILK-Pinch-Parvin complex with integrin adhesion sites. Because of the functional relevance of integrin-linked signaling for endothelial cell (EC) biology, we have explored this pathway in Ilk(-/-) embryonic stem (ES) cells differentiated into ECs and vessel-like structures. We have focused in particular on the mechanistic relevance of ILK-Pinch-Parvin complex-related signaling for EC development and tube formation. Our analysis revealed that the formation of vessel-like structures was strongly reduced in Ilk(-/-) ES cells and that this phenotype could be rescued by re-expression of ILK in ES cells. ECs were MACS sorted from wild-type (WT) and Ilk(-/-) ES cells and functional analysis using intracellular calcium imaging as the read-out yielded a complete lack of vascular endothelial growth factor- and epidermal growth factor-dependent responses. The possibility of a caveolin 1-related defect was investigated by transfecting WT and Ilk(-/-) ECs with a caveolin 1-EGFP fusion protein. Time-lapse microscopy showed that the prominent phenotype is due to altered dynamics of caveolin 1 and to a lack of positioning of caveolin 1 in the vicinity of the plasma membrane and that it is rescued by re-expressing ILK in the Ilk(-/-) ES cells. We also found that the defect is caused by the perturbed organization of microtubules and cortical actin filaments. Thus, ILK is required as a scaffold to allow actin-microtubule interactions and correct positioning of caveolin 1 close to the plasma membrane. This is crucial for signaling compartmentalization in ECs and explains the key role of ILK for EC development and function.

Highly angiogenic CXCR4+CD31+ monocyte subset derived from 3D culture of human peripheral blood

Ex vivo expansion of human circulating angiogenic cells is a major challenge in autologous cell therapy for ischemic diseases. Here, we demonstrate that hematosphere-derived CXCR4+CD31+ myeloid cells using peripheral blood possess robust proangiogenic capacity such as formation of vessel-like structures and tip cell-like morphology in Matrigel. We also found that CD31 positive myeloid cells are principal cellular component of hematospheres by magnetic cell sorting. Flow cytometry analysis showed that fresh peripheral blood contained 40.3 ± 15.2% of CXCR4+CD31+ myeloid cells, but at day 5 of hematosphere culture, most of myeloid cells were CXCR4+CD31+ by 86.9 ± 5.4%. Hematosphere culture significantly increased the production of angiogenic niche-supporting cytokines. Moreover, CD31-homophilic interaction and VEGF–VEGF receptor loop signaling were essential for sphere formation and acquisition of angiogenic capacity in hematospheres. Matrigel plug and ischemic hindlimb model provide in vivo evidence that hematosphere-derived myeloid cells have highly vasculogenic capacities, participate in new and mature vessel formation, and exert therapeutic effects on ischemic hindlimb. In conclusion, our strategy for ex vivo expansion of human CXCR4+CD31+ angiogenic cells using hematospheres provides an autologous therapeutic cell source for ischemic diseases and a new model for investigating the microenvironment of angiogenesis.

Adipose-derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co-culture with adipose-derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co-cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase-14. Moreover, we showed OEC/ASC-induced vessel-like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co-culture-induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system.

Direct‐Write Assembly of 3D Silk/Hydroxyapatite Scaffolds for Bone Co‐Cultures

3D silk/HA microperiodic scaffolds for bone tissue engineering and angiogenesis are fabricated by direct-write assembly. This approach can be used to control filament and spacing size in the scaffold to allow investigation of the effect of scaffold architecture on osteogenesis and vessel-like structure formation from stem cells and endothelial cells.

Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture

The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood. We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches. Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation. Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Ex vivo generation of a highly potent population of circulating angiogenic cells using a collagen matrix

Biomaterials that have the ability to augment angiogenesis are highly sought-after for applications in regenerative medicine, particularly for revascularization of ischemic and infarcted tissue. We evaluated the culture of human circulating angiogenic cells (CAC) on collagen type I-based matrices, and compared this to traditional selective-adhesion cultures on fibronectin. Culture on a collagen matrix supported the proliferation of CD133+ and CD34+CD133+ CACs. When subjected to serum starvation, the matrix conferred a resistance to cell death for CD34+ and CD133+ progenitors and increased phosphorylation of Akt. After 4days of culture, phenotypically enriched populations of endothelial cells (CD31+CD144+) and progenitor cells (CD34+CD133+) emerged. Culture on matrix upregulated the phosphorylation and activation of ERK1/2 pathway members, and matrix-cultured cells also had an enhanced functional capacity for adhesion and invasion. These functional improvements were abrogated when cultured in the presence of ERK inhibitors. The formation of vessel-like structures in an angiogenesis assay was augmented with matrix-cultured cells, which were also more likely to physically associate with such structures compared to CACs taken from culture on fibronectin. In vivo, treatment with matrix-cultured cells increased the size and density of arterioles, and was superior at restoring perfusion in a mouse model of hindlimb ischemia, compared to fibronectin-cultured cell treatment. This work suggests that a collagen-based matrix, as a novel substrate for CAC culture, possesses the ability to enrich endothelial and angiogenic populations and lead to clinically relevant functional enhancements.

Transplantation of Liver Organoids in the Omentum and Kidney

Liver organoids were reconstructed by mouse-immortalized hepatocytes and nonparenchymal cells (sinusoidal endothelial cells and hepatic stellate cells) in a radial-flow bioreactor (RFB). A biodegradable apatite-fiber scaffold (AFS) was used as a scaffold packed in the RFB, which enables three-dimensional cell cultures. The organoids cocultured in the RFB showed a liver-like structure with high-density layers of hepatocytes and the formation of vessel-like structures. A liver organoid consisting of three cocultured cells was transplanted under the kidney capsule (kidney group) or into the omentum (omentum group) using BALB/c nude mice. Transplanted liver organoids survived in the kidney or omentum. The expression of mRNAs of albumin, connexin 26 and 32, hepatocyte nuclear factor 4α, and glucose-6-phosphatase was increased in both groups at 8 weeks after transplantation in comparison to the pretransplant status. Tyrosine aminotransferase appeared only in the omentum group. The results suggested that the functions of liver organoids differed depending on the transplanted site in the recipient animals.

Divergent roles of prokineticin receptors in the endothelial cells: angiogenesis and fenestration

Prokineticins are secreted peptides that activate two G protein-coupled receptors: PKR1 and PKR2. Prokineticins induce angiogenesis and fenestration, but the cognate receptors involved in these functions are unknown. We hypothesized a role for prokineticin receptor signaling pathways and expression profiles in determining the selective effects of prokineticins on coronary endothelial cells (H5V). Activation of the PKR1/MAPK/Akt signaling pathway stimulates proliferation, migration, and angiogenesis in H5V cells, in which PKR1 predominates over PKR2. PKR1 was colocalized with Galpha(11) and was internalized following the stimulation of these cells with prokineticin-2. Knock down of PKR1 or Galpha(11) expression in H5V cells effectively inhibited prokineticin-2-induced vessel formation and MAPK/Akt activation, indicating a role for PKR1/Galpha(11) in this process. However, in conditions in which PKR2 predominated over PKR1, these cells displayed a fenestrated endothelial cell phenotype. H5V cells overexpressing PKR2 displayed large numbers of multivesicular bodies and caveolar clusters and a disruption of the distribution of zonula occluden-1 tight junction protein. Prokineticin-2 induced the colocalization of PKR2 with Galpha(12), and activated Galpha(12), which bound to zonula occluden-1 to trigger the degradation of this protein in these cells. Prokineticin-2 induced the formation of vessel-like structures by human aortic endothelial cells expressing only PKR1, and disorganized the tight junctions in human hepatic sinusoidal endothelial cells expressing only PKR2, confirming the divergent roles of these receptors. Our findings show the functional characteristics of coronary endothelial cells depend on the expression of PKR1 and PKR2 levels and the divergent signaling pathways used by these receptors.

Potency of hair follicle bulb cells implanted into collagen/chitosan porous scaffolds to induce the growth of hair and formation of vessel-like structure in scaffolds

Objective To investigate the possibility of hair follicle reformation induced by hair follicle bulb cells implanted into collagen/chitosan porous scaffolds in vivo, and to observe the angiogenesis in implanted scaffolds.Methods Hair follicle bulb cells obtained by enzyme digestion from the hack skin of C57BL/6J mice were implanted into collagen/chitosan porous scaffolds followed by 2-week organotypie culture.Then,these collagen/chitosan porous scaffolds were transplanted subcutaneously into the dorsal skin of nude mice.Those nude mice transplanted with empty collagen/chitosan porous scaffolds served as the controls.The growth of hair was observed with naked eyes.Six weeks after the transplantation,skin samples were obtained from the recipient site and subjected to histological examination.Results Five weeks after the transplantation,hair growth was observed in the dorsal skin of nude mice.Six weeks later,histological examination revealed fully differentiated hair follicles and vessel-like structures in the center of collagen/chitosan porous scaffolds.However,the transplantation with empty collagen/chitosan porous scaffolds failed to elicit the same response.No hair or follicles were observed in the control mice alpng with small number of vessel-1ike structures.Con-clusions Hair follicle bulb ceils implanted into collagen/chitosan porous scaffolds in vivo could induce hair follicle reformation and promote the formation of vessel-like structure in the scafffold center. Key words: Hair follicle; Collagen; Chitosan; Blood vessels

Differentiation potential of human pancreatic stem cells for epithelial- and endothelial-like cell types

Pancreatic stem cells (PSC) have proved their high plasticity by differentiating into cell types of all three germ layers after the formation of organoid bodies. Motivated by this high differentiation potential this study focused on the immanent stem cell, endothelial and epithelial characteristics of PSC to elucidate whether PSC are a possible source for a stem cell-based in vitro model for screening of pharmaceutical substances. Furthermore, it was investigated whether marker expression was influenced by application of protocols for inducing endothelial or epithelial differentiation originating from research with mesenchymal stem cells or by cultivation on extracellular matrices (ECM). PSC showed expression of relevant stem cell markers CD 45, nestin, Oct 4 and c-kit. As endothelial characteristics they displayed the markers endoglin, VCAM 1, VEGFR 1 and vWF as well as the formation of vessel-like structures. Those endothelial properties were not further intensified by application of protocols employing vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF) and endothelial cell growth medium. As typical epithelial characteristics PSC showed expression of keratin 7, 8, 18 and 19, ZO-1 and catenin beta1. In order to induce epithelial differentiation PSC were cocultured with the lung epithelial cell lines A549 and Calu-3 either by the usage of conditioned medium or by cultivation of PSC on fixed epithelial cells. Depending on the applied coculture system and epithelial cell type used expression of the epithelial marker cadherin-1 was altered compared to control. Cultivation on different ECM changed expression of all investigated markers only marginally. These results confirm that PSC possess endothelial and epithelial properties. Furthermore, epithelial characteristics represented by expression of CDH 1 were altered by coculture with epithelial cell lines.

Transforming growth factor-beta 1 (tgf-β1) induces angiogenesis through vascular endothelial growth factor (vegf)-mediated apoptosis

VEGF and TGF-[beta]1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF-[beta]1 induces apoptosis. We have previously shown that VEGF / VEGF receptor-2 (flk-1) signaling mediates TGF-[beta]1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF-mediated apoptosis is required for TGF-[beta]1 induction of angiogenesis. In vitro the apoptotic effect of TGF-[beta]1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF-[beta]1. Inhibition of VEGF / flk-1 signaling abrogates formation of vessel-like structures by TGF-[beta]1 with an effect comparable to that of z-VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF-[beta]1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF-[beta]1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF-[beta]1 induction of both apoptosis and angiogenesis, an effect similar to that of z-VAD. Thus, TGF-[beta]1 induction of angiogenesis requires rapid and transient endothelial cell apoptosis mediated by VEGF/flk-1. This novel, unexpected role of VEGF and flk-1 indicates VEGF-mediated apoptosis as a potential target to control angiogenesis.

Transforming growth factor-beta 1 (tgf-&amp;946;1) induces angiogenesis through vascular endothelial growth factor (vegf)-mediated apoptosis

AbstractVEGF and TGF-[beta]1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF-[beta]1 induces apoptosis. We have previously shown that VEGF / VEGF receptor-2 (flk-1) signaling mediates TGF-[beta]1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF-mediated apoptosis is required for TGF-[beta]1 induction of angiogenesis. In vitro the apoptotic effect of TGF-[beta]1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF-[beta]1. Inhibition of VEGF / flk-1 signaling abrogates formation of vessel-like structures by TGF-[beta]1 with an effect comparable to that of z-VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF-[beta]1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF-[beta]1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF-[beta]1 induction of both apoptosis and angiogenesis, an effect similar to that of z-VAD. Thus, TGF-[beta]1 induction of angiogenesis requires rapid and transient endothelial cell apoptosis mediated by VEGF/flk-1. This novel, unexpected role of VEGF and flk-1 indicates VEGF-mediated apoptosis as a potential target to control angiogenesis.