Search

Aims: To explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration and the possibility of cotransplantation hUCMSCs and endothelial cells (ECs) for angiogenesis and pulp regeneration in vivo., Materials and Methods: hUCMSCs and human umbilical vein endothelial cells (HUVECs) were cocultured for matrigel angiogenesis assay in vitro and Matrigel plug assay in vivo. Next, we used the transwell coculture system to coculture hUCMSCs and HUVECs in vitro for RNA- sequencing (RNA-seq). Last, encapsulated hUCMSCs and HUVECs in scaffolds were injected into the root segments, and transplanted into immunodeficient mice for dental pulp regeneration., Key Findings: In vitro Matrigel angiogenesis assay and in vivo Matrigel plug assay indicated that cocultured hUCMSCs and HUVECs promote vascular formation of HUVECs, especially in 1:5 (hUCMSCs:HUVECs) coculture group. The RNA-seq result indicated that cocultured HUVECs exhibited high Hif-1 signaling pathway activity. We performed the cell transfection assay to knock down HIF1A-AS2 in HUVECs and then coculture with hUCMSCs, and the expression of VEGFA, HIF1A and PECAM1 were reduced. In pulp regeneration assay, Cotransplantation of hUCMSCs and HUVECs (1,5) group showed pulp-like tissue regeneration., Significance: Cocultured hUCMSCs and HUVECs can promote vascular formation of HUVECs, and the optimal coculture ration is 1:5 (hUCMSCs:HUVECs). hUCMSCs promote angiogenesis of HUVECs through the long noncoding RNA HIF1A-AS2-activation of the Hif-1 signaling pathway. Cotransplantation of hUCMSCs and HUVECs can regenerate dental pulp-like tissue in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia., Methods and Results: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography., Conclusion: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Background: Hemangioma (Hem) is a benign tumor commonly seen in infancy with a relative high morbidity. Human umbilical vein endothelial cell (HUVEC)-derived extracellular vesicles (EVs) are actively participated in Hem. Therefore, this study is designed to figure out the underlying mechanism of HUVEC-derived EVs in Hem., Methods: Initially, EVs were separated from HUVECs and identified. HUVEC-derived EVs in normoxia or hypoxia were then cultivated with Hem endothelial cells (HemECs) to test the proliferation, apoptosis, and migration of HemECs. Microarray analysis was performed to select microRNAs (miRs) with differential expression. miR-210 in hypoxia-induced HUVECs was silenced, and the relevant EVs were extracted and then co-cultured with HemECs to perform biological effect experiments. Then, the target relation between miR-210 and homeobox A9 (HOXA9) was identified by the dual luciferase reporter gene assay and RNA immunoprecipitation assay. Moreover, xenograft transplantation was also applied to confirm the in vitro experiments., Results: Hypoxia-induced HUVECs promoted release of EVs, which were absorbed by HemECs. Hypoxia-induced HUVEC-EVs promoted HemEC proliferation and migration and inhibited apoptosis. miR-210 from the hypoxia-induced HUVEC-EVs was highly expressed and promoted HemEC growth. Silencing miR-210 expression in the hypoxia-induced HUVEC-EVs suppresses Hem development in vivo. In addition, miR-210 targeted HOXA9., Conclusion: Silencing miR-210 in HUVEC-derived EVs could suppress Hem by targeting HOXA9. This investigation may provide novel insights for Hem treatment., (© 2020 S. Karger AG, Basel.)

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal-endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal-endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly ( P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies.-Inglis, S., Kanczler, J. M., Oreffo, R. O. C. 3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche.

Rationale: Endothelial colony forming cells (ECFCs) or late blood outgrowth endothelial cells can be isolated from human cord or peripheral blood, display properties of endothelial progenitors, home into ischemic tissues and support neovascularization in ischemic disease models., Objective: To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation., Methods and Results: We show by transcriptomic analysis that ECFCs are distinguished from endothelial cells of the vessel wall by production of high amounts of CYTL1. Modulation of expression demonstrates that the factor confers increased angiogenic sprouting capabilities to ECFCs and can also trigger sprouting of mature endothelial cells. The data further display that CYTL1 can be induced by hypoxia and that it functions largely independent of VEGF-A (vascular endothelial growth factor-A). By recombinant production of CYTL1 we confirm that the peptide is indeed a strong proangiogenic factor and induces sprouting in cellular assays and functional vessel formation in animal models comparable to VEGF-A. Mass spectroscopy corroborates that CYTL1 is specifically O-glycosylated on 2 neighboring threonines in the C-terminal part and this modification is important for its proangiogenic bioactivity. Further analyses show that the factor does not upregulate proinflammatory genes and strongly induces several metallothionein genes encoding anti-inflammatory and antiapoptotic proteins., Conclusions: We conclude that CYTL1 can mediate proangiogenic functions ascribed to endothelial progenitors such as ECFCs in vivo and may be a candidate to support vessel formation and tissue regeneration in ischemic pathologies.

The aim of this study was to develop an effective cell sheet translocation method using a cell adhesive and temperature-responsive hydroxybutyl chitosan hydrogel (HBC). The polydopamine (PD)-coated HBC hydrogels were prepared by the dopamine self-polymerization on the surface of HBC hydrogel with different coating time, termed as P30, P60 and P120, respectively. Gelling property of HBC was not affected by PD coating. The PD-coated HBC hydrogels promoted the attachment and proliferation of mouse fibroblast cells (L929) and human umbilical vein endothelial cells (HUVECs), and allowed formation of monolayer cell sheet. In vitro translocation of HUVECs sheet could be obtained successively through phase transition of PD coated HBC hydrogel from gel to sol, and the cells sheet transferred from P30 hydrogel to a round cell coverglass maintained relatively complete monolayer and normal cell morphology. The results showed that P30 hydrogel has the potential to be used for cell transplantation therapy., (Copyright © 2018. Published by Elsevier B.V.)

Vascular malformations are defects caused by the abnormal growth of the vasculature. Among them, venous malformation (VM) is an anomaly characterized by slow-flow vascular lesions with abnormally shaped veins, typically in sponge-like configuration. VMs can expand over years causing disfigurement, obstruction of vital structures, thrombosis, bleeding, and pain. Treatments have been very limited and primarily based on supportive care, compression garments, sclerotherapy, and/or surgical resection. Sirolimus treatment has recently shown efficacy in some patients with complicated vascular anomalies, including VMs. Activating somatic TIE2 gene mutations have been identified in up to 60% of VMs and PIK3CA mutations have been found in another 25%. Here, we report a xenograft model of VM that reflects the patients' mutation heterogeneity. First, we established a protocol to isolate and expand in culture endothelial cells (VM-EC) from VM tissue or VM blood of nine patients. In these cells, we identified somatic mutations of TIE2, PIK3CA, or a combination of both. Both TIE2 and PIK3CA mutations induced constitutive AKT activation, while TIE2 mutations also showed high MAPK-ERK signaling. Finally, VM-EC implanted into immune-deficient mice generated lesions with ectatic blood-filled channels with scarce smooth muscle cell coverage, similar to patients' VM. This VM xenograft model could be instrumental to test the therapeutic efficacy of Sirolimus in the presence of the different TIE2 or PIK3CA mutations or to test for efficacy of additional compounds in targeting the specific mutated protein(s), thus enabling development of personalized treatment options for VM patients.

Therapy targeting the neurovascular unit may provide effective neuroprotection against neonatal hypoxia-ischemia (HI). We hypothesized that the peripheral injection of hypoxia-preconditioned human umbilical vein endothelial cells (HUVECs) following HI protects against neurovascular damage and provides long-term neuroprotection in a postpartum (P) day-7 rat pup model. Compared with normoxic HUVECs, hypoxic HUVECs showed enhanced migration and angiogenesis in vitro and had augmented migration effects into the brain when administered intraperitoneally in vivo after HI. Moreover, 24 and 72 h post-HI, the hypoxic HUVECs group but not the normoxic HUVECs or culture-medium groups had significantly higher preservation of microvessels and neurons, and attenuation of blood-brain barrier damage than the normal-saline group. Compared to control or normal-saline groups, only the hypoxic HUVECs group had no impaired foot steps and showed a significant reduction of brain area loss at P42. Next-generation sequencing showed hypoxia-induced upregulation and downregulation of 209 and 215 genes in HUVECs, respectively. Upstream regulator analysis by ingenuity pathway analysis (IPA) identified hypoxia-inducible factor 1-alpha as the key predicted activated transcription regulator. After hypoxia, 12 genes (ADAMTS1, EFNA1, HIF1A, LOX, MEOX2, SELE, VEGFA, VEGFC, CX3CL1, HMMR, SDC, and SERPINE) associated with migration and/or angiogenesis were regulated in HUVECs. In addition, 6 genes (VEGFA, VEGFC, NTN4, TGFA, SERPINE1, and CX3CL1) involved in the survival of endothelial and neuronal cells were also markedly altered in hypoxic HUVECs. Thus, cell therapy by using hypoxic HUVECs that enhance migration and neurovascular protection may provide an effective therapeutic strategy for treating neonatal asphyxia.

Mitochondria contribute to key processes of cellular function, while mitochondrial dysfunction is implicated in metabolic disorders, neurodegenerative diseases, and cardiovascular diseases, in which angiogenesis - the formation of new blood capillaries - is dysregulated. The Hippo signaling transducer, Yes-associated protein (YAP1) binds to the TEA domain (TEAD1) transcription factor and controls angiogenesis. YAP1 also regulates glucose metabolism through peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α), a major player controlling mitochondrial biogenesis. However, the role of YAP1-TEAD1-PGC1α signaling in mitochondrial structure, cellular metabolism, and angiogenesis in endothelial cells (ECs) remains unclear. We now find that knockdown of TEAD1 decreases the expression of PGC1α and suppresses mitochondrial biogenesis, glycolysis, and oxygen consumption in ECs. A YAP1 mutant construct, YAP1S127A, which stimulates binding of YAP1 to TEAD1, upregulates the expression of PGC1α, induces mitochondrial biogenesis, and increases oxygen consumption and glycolytic flux in ECs; in contrast, YAP1S94A, which fails to bind to TEAD1, attenuates these effects. PGC1α knockdown inhibits YAP1S127A-induced EC sprouting in vitro and vascular morphogenesis in the fibrin gel subcutaneously implanted on mice, while overexpression of PGC1α reverses vascular morphogenesis suppressed by YAP1S94A. These results suggest that YAP1-TEAD1 signaling induces mitochondrial biogenesis in ECs and stimulates angiogenesis through PGC1α. Modulation of YAP1-TEAD1-PGC1α signaling in ECs may provide a novel intervention for angiogenesis-related diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin ® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.

Small-diameter vascular grafts are needed for the treatment of coronary artery diseases in the case of limited accessibility of the autologous vessels. Synthetic scaffolds have many disadvantages so in recent years vascular constructs (VCs) made from cellularized natural scaffolds was seen to be very promising but number of studies comprising this area is very limited. In our study, our aim is to generate fully natural triple-layered VC that constitutes all the layers of blood vessel with vascular cells. CD146+ perivascular cells (PCs) were isolated from human umbilical cord vein (HUCV) and differentiated into smooth muscle cells (SMCs) and fibroblasts. They were then combined with collagen type I/elastin/dermatan sulfate and collagen type I/fibrin to form tunica media and tunica adventitia respectively. HUCV endothelial cells (ECs) were seeded on the construct by cell sheet engineering method after fibronectin and heparin coating. Characterization of the VC was performed by immunolabeling, histochemical staining and electron microscopy (SEM and TEM). Differentiated cells were identified by means of immunofluorescent (IF) labeling. SEM and TEM analysis of VCs revealed the presence of three histologic tunicae. Collagen and elastic fibers were observed within the ECM by histochemical staining. The vascular endothelial growth factor receptor expressing ECs in tunica intima; α-SMA expressing SMCs in tunica media and; the tenascin expressing fibroblasts in tunica adventitia were detected by IF labeling. In conclusion, by combining natural scaffolds and vascular cells differentiated from CD146+ PCs, VCs can be generated layer by layer. This study will provide a preliminary blood vessel model for generation of fully natural small-diameter vascular grafts., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1788-1798, 2018., (© 2017 Wiley Periodicals, Inc.)

Emerging evidence suggests that vascular endothelial growth facto (VEGF) is important in the treatment of various ischemic and cardiovascular diseases. However, it often suffers from high cost and easy deactivation with a short half-life. Here, we describe a synthetic 2-N, 6-O-sulfated chitosan (26SCS) with a high affinity to VEGF promoting the binding of the signaling protein to its VEGF receptor 2 (VEGFR2), activating receptor phosphorylation and pro-angiogenic related genes expression, and further stimulating downstream VEGF-dependent endothelial cell viability, migration, tube formation and rat aortic rings outgrowth. Interestingly, the obvious recruitment of mural cells were occurred to stabilize the sprouted microvessels. In addition, the pro-angiogenic potential of 26SCS composited VEGF was confirmed in vivo using the chick embryo chorioallantoic membrane (CAM) assay with an extensive perfusable vascular network. A longer monitoring was administered subcutaneously to mice in a biocompatible gelatin sponge and showed that VEGF with 26SCS had the capability to efficiently enhance neovascularization. These findings highlight that 26SCS, the semi-synthetic natural polymer, may be a promising coagent with VEGF for vascular therapy., Statement of Significance: Vascular endothelial growth factor (VEGF) is crucial for facilitating angiogenesis to supply oxygen and nutrient during wound healing and tissue regeneration. However, appropriate use of VEGF is an ongoing challenge due to its rapidly clearance and severe side effects at higher dosage. In this study, we described a synthetic 2-N, 6-O-sulfated chitosan (26SCS) with a high affinity to VEGF, which could significantly promote its binding capacity to VEGF receptor 2 and further stimulate the angiogenic behavior of endothelial cells. We further confirmed that 26SCS was spatially combined with VEGF in a "lying manner", and this spatial arrangement was more conducive to exposure of the receptor binding domain of VEGF. Additionally, it also promoted in vivo angiogenesis in a chicken chorioallantoic membrane assay and mouse subcutaneous implant model. This strategy may afford a new avenue to enhance pro-angiogenic capacity of VEGF., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel., Methods: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5 + cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis., Results: The resultant hPSC-derived CDH5 + cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months., Conclusions: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs., (© 2017 American Heart Association, Inc.)

A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.

Biomaterials are only used as carriers of cells in the conventional tissue engineering. Considering the multi-cell environment and active cell-biomaterial interactions in tissue regeneration process, in this study, structural signals of aligned electrospun nanofibers and chemical signals of bioglass (BG) ionic products in cell culture medium are simultaneously applied to activate fibroblast-endothelial co-cultured cells in order to obtain an improved skin tissue engineering construct. Results demonstrate that the combined biomaterial signals synergistically activate fibroblast-endothelial co-culture skin tissue engineering constructs through promotion of paracrine effects and stimulation of gap junctional communication between cells, which results in enhanced vascularization and extracellular matrix protein synthesis in the constructs. Structural signals of aligned electrospun nanofibers play an important role in stimulating both of paracrine and gap junctional communication while chemical signals of BG ionic products mainly enhance paracrine effects. In vivo experiments reveal that the activated skin tissue engineering constructs significantly enhance wound healing as compared to control. This study indicates the advantages of synergistic effects between different bioactive signals of biomaterials can be taken to activate communication between different types of cells for obtaining tissue engineering constructs with improved functions., Statement of Significance: Tissue engineering can regenerate or replace tissue or organs through combining cells, biomaterials and growth factors. Normally, for repairing a specific tissue, only one type of cells, one kind of biomaterials, and specific growth factors are used to support cell growth. In this study, we proposed a novel tissue engineering approach by simply using co-cultured cells and combined biomaterial signals. Using a skin tissue engineering model, we successfully proved that the combined biomaterial signals such as surface nanostructures and bioactive ions could synergistically stimulate the cell-cell communication in co-culture system through paracrine effects and gap junction activation, and regulated expression of growth factors and extracellular matrix proteins, resulting in an activated tissue engineering constructs that significantly enhanced skin regeneration., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background and Objective: Chronic periodontitis causes damage to tooth-supporting tissues, resulting in tooth loss in adults. Recently, cell-sheet-based approaches have been studied to overcome the limitations of conventional cytotherapeutic procedures for periodontal regeneration. The purpose of the present study was to investigate the regenerative potential of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs) in three-dimensional (3D) cell sheet constructs for periodontal regeneration in vivo., Material and Methods: PDLSCs, HUVECs or co-cultures of both cells were seeded onto temperature-responsive culture dishes, and intact cell sheets were fabricated. Cell sheets were wrapped around the prepared human roots in three different combinations and implanted subcutaneously into immunodeficient mice., Results: Histological evaluation revealed that after 2, 4 and 8 wk of implantation, periodontal ligament-like tissue arrangements were observed around the implanted roots in experimental groups compared with controls. Vascular lumens were also observed in periodontal compartments of HUVEC-containing groups. Periodontal ligament regeneration, cementogenesis and osteogenesis were evident in the experimental groups at both weeks 4 and 8, as shown by immunostaining for periostin and bone sialoprotein. Human cells in the transplanted cell sheets were stained by immunohistochemistry for the presence of human mitochondria., Conclusions: The 3D cell sheet-based approach may be potentially beneficial and is thus encouraged for future regenerative periodontal therapy., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.

Objective: CD98 regulates integrin signaling and is critical for tumor cell proliferation. It is also expressed on endothelial cells (EC), but its role in angiogenesis is unclear., Approach and Results: We used specific genetic targeting and antibody blockade approaches to examine the function of CD98 in EC proliferation, blood vessel growth, and tumor angiogenesis. It is upregulated on angiogenic ECs, and EC-specific deletion of CD98 in mice inhibited tumor growth, retinal angiogenesis, and EC proliferation. Reconstitution with CD98 mutants showed that integrin and CD98 interaction is necessary for EC survival and growth. Moreover, anti-CD98 treatment inhibited vessel formation and reversed EC-assisted tumor growth., Conclusions: Our findings demonstrate a requirement for CD98 in EC growth and suggest that CD98-specific reagents could have a dual anticancer effect: directly by inhibiting tumor cell proliferation and indirectly by preventing tumor angiogenesis., (© 2016 American Heart Association, Inc.)

Vascular-derived endothelial cell (EC) network prefabrication in three-dimensional (3D) tissue constructs before transplantation is useful for inducing functional anastomosis with the host vasculature. However, the clinical application of ECs is limited by cell isolation from the existing vasculature, because of the requirement for invasive biopsies and difficulty in obtaining a sufficient number of cells. Endothelial colony-forming cells (ECFCs), which are a subtype of endothelial progenitor cells in the blood, have a strong proliferative and vasculogenic potential. This study attempted to fabricate prevascular 3D cell-dense tissue constructs using cord blood-derived ECFCs and evaluate the in vivo angiogenic potential of these constructs. Human umbilical vascular endothelial cells (HUVECs) were also used in comparison with ECFCs, which were sandwiched between two human dermal-derived fibroblast (FB) sheets using a fibrin-coated cell-sheet manipulator. The inserted ECFCs in double-layered FB sheets were cultured for 3 days, resulting in the formation of network structures similar to those of HUVECs. Additionally, when ECFCs were sandwiched with three FB sheets, a lumen structure was found in the triple-layered cell-sheet constructs at 3 days after co-culture. These constructs containing ECFCs were transplanted into the subcutaneous tissue of immune-deficient rats. One week after transplantation, ECFC-lined functional microvessels containing rat erythrocytes were observed in the same manner as transplanted HUVEC-positive grafts. These results suggest that ECFCs might become an alternative cell source for fabricating a prevascular structure in 3D cell-dense tissue constructs for clinical application. Copyright © 2013 John Wiley & Sons, Ltd., (Copyright © 2013 John Wiley & Sons, Ltd.)

The present study used a previously developed three-dimensional Gelatin/Hydroxyapatite (Gel/HA) homogeneous nanocomposite scaffold with porosity of 82% and interconnecting pores ranging from 300 to 500 μm. Cell-seeded scaffolds were used to evaluate bone regeneration of rat critical-size calvarial defect. Totally, 36 male Wistar rats were randomly divided into four experimental groups, including blank defect (defects without any graft), blank scaffold (defects filled with Gel/HA scaffold without cells), and two groups of cell-seeded scaffolds (defects filled with either Gel/HA scaffold seeded with osteoblast-like and endothelial cells or osteoblast-like cell-seeded constructs). After 1, 4, and 12 weeks of scaffold implantation, rats were sacrificed and the calvaria were harvested for histological, immunohistochemical and histomorphometric analysis. In vitro tests showed that scaffolds were nontoxic to cells and promoted ideal cellular attachment. In vivo investigation on scaffold revealed that blank calvarial defects indicated incomplete tissue coverage and little evidence of bone healing. However, blank scaffold and cell-seeded scaffolds significantly promoted osteoconduction and ostegogenesis. Taken together, pre-seeded Gel/HA nanocomposite scaffold with osteoblasts and endothelial cells presented an effective combination to improve osteogenesis in the engineered bone implant. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1770-1778, 2016., (© 2016 Wiley Periodicals, Inc.)

There are increasing numbers of patients underwent partial nephrectomy, and recovery of disturbed renal function is imperative post partial nephrectomy. We previously have demonstrated the decellularized (DC) scaffolds could mediate the residual kidney regeneration and thus improve disturbed renal function after partial nephrectomy. However, the cellular changes including the angiogenesis in the implanted DC scaffold has not yet been elaborated. In this study, we observed that the scaffold promoted the proliferation of human umbilical vein endothelial cells (HUVEC) that adhered to the DC scaffold in vitro. We next examined the pathological changes of the implanted DC graft in vivo, and found a decreased volume of the scaffold and a dramatic angiogenesis within the scaffold. The average microvessel density (aMVD) increased at the early stage, while decreased at the later stage post transplantation. Expression level of vascular endothelial growth factor (VEGF) showed similar dynamic changes. In addition, many endothelial cells (ECs) and endothelial progenitor cells (EPCs) were distributed in the region which contained active angiogenesis in the scaffold. However, the implanted graft became fibrosis and the angiogenesis degraded at final stage roughly 8 weeks post transplantation. Our data indicate that DC scaffold can be vascularized in vivo and possible mechanisms are discussed., Competing Interests: The authors declare no competing financial interests.

Cell-based therapy has been studied as an attractive strategy for therapeutic angiogenesis. However, obtaining a stable vascular structure remains a challenge due to the poor interaction of transplanted cells with native tissue and the difficulty in selecting the optimal cell source. In this study, we developed a cell patch of cocultured human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) using thermosensitive hydrogels for regeneration of mature vasculatures. In vitro characterization of HUVECs in the cocultured group revealed the formation of a mesh-like morphology over 5 days of culture. Vascular endothelial growth factor expression was also upregulated in the cocultured group compared with HUVECs only. The cell patch seeded with HUVECs, SMCs, or both cell type was prepared on the synthetic thermosensitive and cell interactive hydrogels, and readily detached from the hydrogel within 10 min by expansion of the hydrogel when the temperature was decreased to 4°C. We then investigated the therapeutic effect of the cell patch using a hind limb ischemic model of an athymic mouse. Overall, the group that received a cell patch of cocultured HUVECs and SMCs had a significantly retarded rate of necrosis with a significant increase in the number of arterioles and capillaries for 4 weeks compared with the groups transplanted with only HUVECs or SMCs. Dual staining of smooth muscle alpha actin and human nuclear antigen showed that the implanted cell patch was partially involved in vessel formation. In summary, the simple transplantation of a cocultured cell patch using a hydrogel system could enhance therapeutic angiogenesis through the regeneration of matured vascular structures.

The aim of this study was to compare the effect of implantation site (i.e., subcutaneous, SQ vs. intramuscular, IM) on bone forming capacity of cell-based and growth factor-based scaffolds in athymic nude rats after an implantation period of 8 weeks. Cell-based scaffolds consisted of porous hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds seeded with either human adipose tissue-derived mesenchymal stem cells (AT-MSCs) only or both AT-MSCs and human umbilical vein endothelial cells (HUVECs), which were precultured in osteogenic medium for 7 days. Growth factor-based scaffolds consisted of porous HA/TCP scaffolds with 20 µg preadsorbed bone morphogenetic protein-2 (BMP-2). Histological and histomorphometrical analysis were used to assess bone formation. A differentiation experiment was performed in parallel to compare the in vitro osteogenic capacity of cell-based scaffolds. The results showed that cell-based scaffolds showed evident osteogenic differentiation in vitro, with only marginal differences between AT-MSCs only and AT-MSCs/HUVECs. In vivo, none of the cell-based scaffolds showed bone formation, irrespective of the site of implantation. In contrast, all growth factor-based scaffolds showed bone formation at both implantation sites without differences in the amount of formed bone. In conclusion, the results of this study demonstrated that the bone forming capacity of HA/TCP scaffolds with pre-adsorbed BMP-2 was equal at different ectopic implantation sites. Further, despite obvious in vitro osteogenic differentiation of AT-MSCs and AT-MSCS/HUVECs on HA/TCP scaffolds, no bone formation of these cell-based scaffolds was observed in vivo. This indicates further investigation on bone formation mechanisms of AT-MSCs is needed before AT-MSCs can be used as a cytotherapeutic treatment in clinics., (© 2014 Wiley Periodicals, Inc.)

Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.

Volume stability and growth of tissue engineered adipose tissue equivalents using adipose-derived stem cells (ASCs) rely strongly on angiogenesis and neovascularization to support the maintenance of cells. An attractive cellular approach is based on coimplantation of endothelial cells to create a vascular network. Endothelial progenitor cells (EPCs) are a promising cell population, since they can be easily isolated from autologous human peripheral blood and thus represent a clinically feasible option. We have previously shown in in vitro and semi-in vivo studies that ASCs exert beneficial effects on EPCs in terms of enhanced tube formation and formation of blood vessels, respectively. In this study, we investigated the in vivo effects of coimplantation on endothelial cell-mediated neovascularization and ASC-mediated adipose tissue formation. For this purpose, human ASCs and human EPCs (or HUVECs as direct comparison to EPCs) were suspended alone or in coculture in fibrin and subcutaneously injected into the back of athymic nude mice and explanted after 1, 3 or 6months. Our results show that monocultures of EPCs or HUVECs were not able to perform vasculogenesis and constructs exhibited complete resorption already after 1month. However, a remarkable difference between EPCs and HUVECs was detected when coimplanted with ASCs. While coimplanted HUVECs gave rise to a stable neovasculature which was characterized by perfusion with erythrocytes, coimplanted EPCs showed no ability to form vascular structures. In the case of HUVEC-derived neovasculature, coimplanted ASCs displayed perivascular properties by stabilizing these neovessels. However, formation of human adipose tissue was independent of coimplanted endothelial cells. Our results indicate that HUVECs are superior to EPCs in terms of promoting in vivo neovascularization and recruiting perivascular cells for vessel stabilization when coimplanted with ASCs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell sheets harvested from temperature-responsive cell culture dishes (TRDs) has attracted considerable attention as effective tools for reconstructing the lost functions of tissues and organs in the regenerative medicine field. However, because of their thinness, handling problems sometimes arise when transferring cell sheets from a TRD to a target surface. In this study, we developed a facile cell transfer method referred to as in situ gelation by using both gelatin hydrogel and a support membrane. Gelation and low-temperature processes were simultaneously performed on TRD. Confluent cultured cells were efficiently harvested from TRD in less than 5 min by decreasing the incubation temperature to 20°C. Harvested cells were found to maintain their cell viability, extracellular matrix, and original shape, thus allowing transfer of the cells to another surface with a short incubation time at 37°C. This method is applicable for various cell types regardless of the formation of tight cell-cell junctions. In addition, because of the high flexibility of the gelatin-coated membrane, cells were efficiently transferred to the surface of a mouse subcutis and liver. When compared with conventional cell sheet manipulation methods, the interaction between the cell surface and membrane was reinforced by the uniformly formed gelatin gel layer without using a special device. Therefore, the in situ gelation method is a promising technique for cell sheet-based tissue engineering and regenerative medicine., (© 2014 Wiley Periodicals, Inc.)

Background and Purpose: We investigated the neurorestorative effects and underlying mechanisms of stroke treatment with human umbilical cord blood cells (HUCBCs) in Type one diabetes mellitus (T1DM) rats., Methods: Type one diabetes mellitus rats were subjected to middle cerebral artery occlusion (MCAo) and 24 h later were treated with: (1) phosphate-buffered-saline; (2) HUCBCs. Brain endothelial cells (MBECs) were cultured and capillary tube formation was measured., Results: Human umbilical cord blood cells treatment significantly improved functional outcome and promoted white matter (WM) remodeling, as identified by Bielschowsky silver, Luxol fast blue and SMI-31 expression, increased oligodendrocyte progenitor cell and oligodendrocyte density after stroke in T1DM rats. HUCBC also promoted vascular remodeling, evident from enhanced vascular and arterial density and increased artery diameter, and decreased blood-brain barrier leakage. HUCBC treatment also increased Angiopoietin-1 and decreased receptor for advanced glycation end-products (RAGE) expression compared to T1DM-MCAo control. In vitro analysis of MBECs demonstrated that Ang1 inversely regulated RAGE expression. HUCBC and Ang1 significantly increased capillary tube formation and decreased inflammatory factor expression, while anti-Ang1 attenuated HUCBC-induced tube formation and antiinflammatory effects., Conclusion: Human umbilical cord blood cells is an effective neurorestorative therapy in T1DM-MCAo rats and the enhanced vascular and WM remodeling and associated functional recovery after stroke may be attributed to increasing Angiopoietin-1 and decreasing RAGE., (© 2014 John Wiley & Sons Ltd.)

Objective: It is well established that angiogenesis is a complex and coordinated multistep process. However, there remains a lack of information about the genes that regulate individual stages of vessel formation. Here, we aimed to define the role of human interferon-induced transmembrane protein 1 (IFITM1) during blood vessel formation., Approach and Results: We identified IFITM1 in a microarray screen for genes differentially regulated by endothelial cells (ECs) during an in vitro angiogenesis assay and found that IFITM1 expression was strongly induced as ECs sprouted and formed lumens. We showed by immunohistochemistry that human IFITM1 was expressed by stable blood vessels in multiple organs. siRNA-mediated knockdown of IFITM1 expression spared EC sprouting but completely disrupted lumen formation, in both in vitro and in an in vivo xeno-transplant model. ECs lacking IFITM1 underwent early stages of lumenogenesis (ie, intracellular vacuole formation) but failed to mature or expand lumens. Coimmunoprecipitation studies confirmed occludin as an IFITM1 binding partner in ECs, and immunocytochemistry showed a lack of occludin at endothelial tight junctions in the absence of IFITM1. Finally, time-lapse video microscopy revealed that IFITM1 is required for the formation of stable cell-cell contacts during endothelial lumen formation., Conclusions: IFITM1 is essential for the formation of functional blood vessels and stabilizes EC-EC interactions during endothelial lumen formation by regulating tight junction assembly.

Aims: We elucidated the therapeutic potential of human umbilical vein endothelial cells (HUVECs) for ameliorating progressive heart failure in a myocardial infarction (MI) rat model., Main Methods: MI was induced by ligation of left anterior descending artery, and HUVEC was transplanted 1week after MI. Cardiac function was evaluated by echocardiography, and histological analyses were performed., Key Findings: Phosphate-buffered saline (MI-V, n=5) or HUVEC (MI-HV, n=5) were injected into the border zone and infarcted area 7days after ligation of the left coronary artery in rats. The MI-HV group showed attenuation of left ventricular (LV) remodeling compared with the MI-V group. In the infarcted myocardium, a few of injected HUVEC was retained up to 28days. The ratios of matrix metalloproteinase (MMP)-2 or MMP-9 to tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-3 were decreased in the MI-HV group compared with the MI-V group. In vivo zymography analysis showed that HUVEC transplantation decreased the activities of MMP-2 and MMP-9. In immunohistochemistry, decreased MMP-2 and increased TIMP-1 and TIMP-3 expression were observed at 48h after HUVEC transplantation. These effects on MMP/TIMP balance were inhibited by L-NAME administration (an eNOS inhibitor, 10mg/kg). NOS inhibition decreased the protein expressions of TIMP-1 and TIMP-3 but did not change the protein expressions of MMP-2 and MMP-9., Significance: Our data suggest that altered balance between MMP and TIMP by HUVEC transplantation contributed to attenuation of ventricular remodeling after MI via eNOS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although a number of growth factors and receptors are known to control tumor angiogenesis, relatively little is known about the mechanism by which these factors influence the directional endothelial cell migration required for cancer microvessel formation. Recently, it has been shown that the focal adhesion protein paxillin is required for directional migration of fibroblasts in vitro. Here, we show that paxillin knockdown enhances endothelial cell migration in vitro and stimulates angiogenesis during normal development and in response to tumor angiogenic factors in vivo. Paxillin produces these effects by decreasing expression of neuropilin 2 (NRP2). Moreover, soluble factors secreted by tumors that stimulate vascular ingrowth, including vascular endothelial growth factor (VEGF), also decrease endothelial cell expression of paxillin and NRP2, and overexpression of NRP2 reverses these effects. These results suggest that the VEGF-paxillin-NRP2 pathway could represent a new therapeutic target for cancer and other angiogenesis-related diseases.

Cell-based therapeutic neovascularization is a promising method for treating ischemic disorders. In this work, human umbilical vein endothelial cells (HUVECs) were thoroughly premixed with cord-blood mesenchymal stem cells (cbMSCs) and cultivated to form three-dimensional (3D) cell aggregates for cellular cardiomyoplasty. In the in vitro study, tubular networks were formed at day 1 after the co-culturing of dissociated HUVECs and cbMSCs on Matrigel; however, as time progressed, the grown tubular networks regressed severely. Conversely, when 3D cell aggregates were grown on Matrigel, mature and stable tubular networks were observed over time, under the influence of their intensive cell-extracellular matrix (ECM) interactions and cell-cell contacts. 3D cell aggregates were transplanted into the peri-infarct zones of rats with myocardial infarction (MI) via direct intramyocardial injection. Based on our pinhole single photon emission computed tomography (SPECT) myocardial-perfusion observations, echocardiographic heart-function examinations and histological analyses, the engrafted 3D cell aggregates considerably enhanced the vascular densities and the blood flow recovery in the ischemic myocardium over those of their dissociated counterparts, thereby reducing the size of perfusion defects and restoring cardiac function. These results demonstrate that the intramuscular delivery of 3D cell aggregates of HUVECs/cbMSCs can be a valuable cell-based regenerative therapeutic strategy against MI., (© 2013.)

In this study, thermosensitive hydrogels incorporated with multiple cell-interactive factors were developed as a substrate to form monolayer of human umbilical vein endothelial cells (HUVECs) that can be detached and transferrable to target sites as a cell-sheet in response to temperature change. The cell adhesive peptide (RGD) and growth factor (bFGF) covalently incorporated within the hydrogel significantly enhanced adhesion and proliferation of HUVECs, allowing for the formation of their confluent monolayer. Meanwhile, the precisely controllable change in the size of the hydrogels was observed by a repeated increase and decrease in temperature from 37 to 4 °C. By exploiting this unique behavior, the detachment and transfer of HUVEC sheet confluently cultured at 37 °C was rapidly induced within 10 min by expansion of the hydrogels when the temperature was decreased to 4 °C. The transferred cell sheet was highly viable and maintained robust cell-cell junction. Finally, the process of cell sheet transfer was directly applied onto an ischemic injury in the hind limb of mice. The transplanted HUVECs as a sheet retarded tissue necrosis over 14 days in comparison with that of direct injection of the same number of cells. Our results suggest that the developed multifunctional Tetronic-tyramine hydrogels could serve as an ideal substrate to modulate the formation of an endothelial cell layer that could potentially be utilized to treat peripheral arterial disease.

Cell transplantation for therapeutic neovascularization holds great promise for treating ischemic diseases. This work prepared three-dimensional aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) with different levels of internal hypoxia by a methylcellulose hydrogel system. We found that few apoptosis occurred in these cell aggregates, despite developing a hypoxic microenvironment in their inner cores. Via effectively switching on the hypoxia-inducible factor-1α-dependent angiogenic mechanisms, culturing the internally hypoxic HUVEC/cbMSC aggregates on Matrigel resulted in formation of extensive and persistent tubular networks and significant upregulation of pro-angiogenic genes. As the level of internal hypoxia created in cell aggregates increased, the robustness of the tubular structures developed on Matrigel increased, and expression levels of the pro-angiogenic genes also elevated. Transplantation of hypoxic HUVEC/cbMSC aggregates into a mouse model of an ischemic limb significantly promoted formation of functional vessels, improved regional blood perfusion, and attenuated muscle atrophy and bone losses, thereby rescuing tissue degeneration. Notably, their therapeutic efficacy was clearly dependent upon the level of internal hypoxia established in cell aggregates. These analytical results demonstrate that by establishing a hypoxic environment in HUVEC/cbMSC aggregates, their potential for therapeutic neovascularization can be markedly enhanced., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Angiogenesis inhibitors combined with other anticancer drugs have been shown to inhibit tumor growth in animal models and some of them were recently used in clinical trials. In the present study, a whole hepatocellular carcinoma cell lysate based vaccine with diphtheria toxin (DT) and two tandem repeats of microbial HSP70 peptide epitope 407-426 (2 mHSP70407-426, M2) as adjuvant, which was called HDM, was combined with a whole human umbilical vein endothelial cell (HUVEC) vaccine to develop a combination treatment regimen. This combination treatment regimen was named HUVEC-HDM, which was supposed to enhance its antitumor efficiency. HUVEC-HDM was administrated subcutaneously in both prophylactic and therapeutic procedures. Compared to either single vaccine, HUVEC-HDM induced a more significant inhibition on the growth and metastasis of H22 hepatocellular carcinoma in mice and prolonged the survival of tumor-bearing mice. Besides, HUVEC-HDM immunization elicited strong humoral and cellular immune responses targeting tumor cell as well as tumor angiogenesis, which could be responsible for the enhanced antitumor effect. Moreover, histochemistry analysis showed that HUVEC-HDM induced large areas of continuous necrosis within tumors, correlating well with the extent of tumor inhibition. These results not only highlight the superiority of the combined HUVEC-HDM treatment regimen, but also support the translation of such approaches into the clinic for the treatment of patients with hepatocellular carcinoma.

Background: Recently, we showed the angio-vasculogenic potential of uncultured human peripheral blood (hPB)-derived CD31(+) cells. However, thus far, the angiogenic property of the cultured hPB-derived CD31(+) (C-31(+)) cells is unknown. Thus, this study aimed to assess the angiogenic potency of C-31(+) cells on experimental ischemia., Methods: CD31(+) and CD31(-) cells were isolated by magnetic bead separation technique, and cultured in EBM-2 complete medium for 6days. The expression of multiple angiogenic genes in these cells was measured using qRT-PCR. In addition, endothelial progenitor cell culture and matrigel network formation assays were performed. A mouse model of hindlimb ischemia induced by surgical resection of the right femoral artery was used, and the C-31(+) cells were intramuscularly transplanted into the ischemic area. Immunohistochemical analysis was also performed., Results: C-31(+) cells exclusively showed higher colony-forming activity, and gave rise to EPCs. C-31(+) cells also induced higher endothelial network formation, and exhibited higher pro-angiogenic and lower inflammatory gene expression. In our ischemic hindlimb model, transplantation of C-31(+) cells induced increased blood perfusion (0.652±0.03 vs. 0.47±0.04; P<0.01) and increased capillary density (85±5.5 vs. 57±4.1; P<0.01) as compared to C-31(-) cells. In addition, angiogenic factors were markedly upregulated after the transplantation of C-31(+) cells, indicating that C-31(+) cells contributed to the neovascularization., Conclusions: The high angiogenic and therapeutic potential of C-31(+) cells observed in our ischemic animal model suggests a novel role of hPB-derived cultured CD31(+) cells in the treatment of ischemic cardiovascular diseases., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Human blood-derived CD133(+) cell populations, which are believed to represent a hematopoietic/endothelial progenitor fraction, have the ability to promote the repair of injured spinal cord in animal models. However, the mechanisms by which CD133(+) cell transplantation promotes spinal cord regeneration remain to be clarified. Another possible hurdle on the way to clinical applicability of these cells is their scarce representation in the overall population of mononuclear cells. We therefore analyzed and compared ex-vivo expanded human cord blood derived CD133(+) cells with freshly isolated CD133(+) cells as well as corresponding CD133(-) control mononuclear cells in respect to their ability to promote spinal cord repair using in vitro assays and cell transplantation into a mouse spinal cord injury model. In vitro, expanded cells as well as fresh CD133(+) cells formed endothelial progenitor cell (EPC) colonies, whereas CD133(-) cells formed no EPC colonies. In vivo, the administration of fresh CD133(+) and expanded cells enhanced angiogenesis, astrogliosis, axon growth and functional recovery after injury. In contrast, the administration of CD133(-) cells failed to promote axon growth and functional recovery, but moderately enhanced angiogenesis and astrogliosis. In addition, high-dose administration of expanded cells was highly effective in the induction of regenerative processes at the injured spinal cord., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Both adipose-derived stromal cells (ASCs) and endothelial progenitor cells (EPCs) have high potential for promoting tissue revascularization and functional recovery in acute myocardial infarction (AMI) models. We investigated the functional effects of intramyocardial transplantation of a human ASC and EPC mixture in immunodeficient rats after MI., Methods: MI was induced by ligating left anterior descending coronary artery. Survived rats were randomly assigned to 1 of 4 different groups: the control group (n=10, saline in 100μL), the ASC group (n=10, 10(6) ASCs), the EPC group (n=10, 10(6) EPCs), or the ASC+EPC group (n=10, 2×10(5) ASCs+8×10(5) EPCs). Left ventricular (LV) function was compared using echocardiography during the 28-day follow-up. GAP43+ nerve sprouting and smooth muscle α-actin+angiogenesis were also compared., Results: Serial changes in LV ejection fraction (EF) and fractional shortening revealed significant increases in the ASC, EPC, and ASC+EPC groups when compared to the control group during the follow-up (49±3%, 49±4%, 47±4%, 39±2%, P<0.001, respectively for LVEF) (33±4%, 32±2%, 31±2%, 23±2%, P=0.002, respectively for fractional shortening). The number of α-actin+arterioles and GAP43+ nerve area was significantly greater in the ASC, EPC, and ASC+EPC groups when compared to the control group in the peri-infarct area (34.4±1.0/mm(2), 35.9±1.1/mm(2), 35.3±0.9/mm(2), 17.4±0.7/mm(2), P<0.001, respectively for angiogenesis) (346.2±10.7μm(2)/mm(2), 357.2±12.8μm(2)/mm(2), 368.0±9.7μm(2)/mm(2), 174.6±7.9μm(2)/mm(2), P<0.001, respectively for nerve sprouting)., Conclusions: [corrected] Intramyocardial injections of ASCs, EPCs, or ASCs+EPCs are effective modalities for the treatment of myocardial damage in rats and may expand the potential clinical application of ASC or EPC therapy in patients with ischemic heart disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The proximity of cells in three-dimensional (3D) organization maximizes the cell-cell communication and signaling that are critical for cell function. In this study, 3D cell aggregates composed of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) were used for therapeutic neovascularization to rescue tissues from critical limb ischemia. Within the cell aggregates, homogeneously mixed HUVECs and cbMSCs had direct cell-cell contact with expressions of endogenous extracellular matrices and adhesion molecules. Although dissociated HUVECs/cbMSCs initially formed tubular structures on Matrigel, the grown tubular network substantially regressed over time. Conversely, 3D HUVEC/cbMSC aggregates seeded on Matrigel exhibited an extensive tubular network that continued to expand without regression. Immunostaining experiments show that, by differentiating into smooth muscle cell (SMC) lineages, the cbMSCs stabilize the HUVEC-derived tubular network. The real-time PCR analysis results suggest that, through myocardin, TGF-β signaling regulates the differentiation of cbMSCs into SMCs. Transplantation of 3D HUVEC/cbMSC aggregates recovered blood perfusion in a mouse model of hindlimb ischemia more effectively compared to their dissociated counterparts. The experimental results confirm that the transplanted 3D HUVEC/cbMSC aggregates enhanced functional vessel formation within the ischemic limb and protected it from degeneration. The 3D HUVEC/cbMSC aggregates can therefore facilitate the cell-based therapeutic strategies for modulating postnatal neovascularization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human umbilical cord blood cells (HUCBCs) have been employed as a restorative treatment for experimental stroke. In this study, we investigated whether transplantation of sub-therapeutic doses of HUCBCs and Simvastatin enhances cerebral vascular remodeling after stroke. Adult male Wistar rats (n=34) were subjected to transient middle cerebral artery occlusion (MCAo) and treated with: phosphate-buffered solution (PBS, gavaged daily for 7 days); Simvastatin (0.5mg/kg, gavaged daily for 7 days); HUCBCs (1×10(6), injected once via tail vein); and combination Simvasatin with HUCBCs, starting at 24h after MCAo. There was no significant difference between Simvastatin- or HUCBC-monotherapy and MCAo-alone group. Combination treatment 24h post-stroke significantly increased the perimeter of von Willebrand factor (vWF)-positive vessels, the diameter and density of alpha smooth muscle actin (αSMA)-positive arteries, and the percentage of 5-bromodeoxyuridine (BrdU)-positive endothelial cells (ECs) in the ischemic boundary zone (IBZ) compared with MCAo-alone or HUCBC-monotherapy 14 days after MCAo (p<0.05, n=8/group); Combination treatment significantly increased the densities of vWF-vessels and αSMA-arteries as well as the densities of BrdU-ECs and BrdU-positive smooth muscle cells (SMCs) in vascular walls in the IBZ compared with Simvastatin-monotherapy. Moreover, the increased BrdU-ECs and BrdU-SMCs were significantly correlated with neurological functional outcome 14 days after MCAo. Combination treatment also significantly increased the expression of Angiopoietin-1 (Ang1), Tie2 and Occludin in the IBZ (p<0.05, n=8/group). The in vitro experiments showed that combination treatment and Ang1 significantly increased capillary-like tube formation and arterial cell migration; anti-Ang1 significantly reduced combination treatment-induced tube-formation and artery cell migration (p<0.05, n=6/group). These findings indicated that a combination of sub-therapeutic doses of Simvastatin and HUCBCs treatment of stroke increases Ang1/Tie2 and Occludin expression in the ischemic brain, amplifies endogenous angiogenesis and arteriogenesis, and enhances vascular remodeling which in concert may contribute to functional outcome after stroke., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Disruption of blood vessels caused by a spinal cord injury leads to tissue hypoxia. This hypoxic condition reduces the survival of transplanted stem cells, consequentially decreasing the effectiveness of stem cell therapy. In this study, we investigated the correlation between angiogenesis and the survival of transplanted neural stem cells in a spinal cord injury model. Hypoxia-specific luciferase-expressing neural stem cells (EpoSV-Luc NSC) were used as a tool for the detection of hypoxia caused by a spinal cord injury. In vivo, angiogenesis by cotransplantation of endothelial cells quickly recovered tissue hypoxia caused by a spinal cord injury. As a result, cotransplantation of endothelial cells improved the survival of neural stem cells transplanted into the injured spinal cord.

Purpose: Microencapsulated hepatocytes might solve immunological rejection, broadening a new perspective for the treatment of fulminant hepatic failure (FHF). However, the transplantation of microcapsulated hepatocytes is limited by low cell viability. Nevertheless, the co-microencapsulation of hepatocytes and human umbilical vein endothelial cells (HUVECs) may make the treatment of FHF more promising., Methods: We prepared the microcapsules using the high-voltage electrostatic droplet spray method, transplanted the empty microcapsules, isolated hepatocytes, microcapsulated hepatocytes, and co-microencapsulated hepatocytes and HUVEC intraperitoneally into rat models of FHF induced by D-aminogalactose (D-gal). After 1, 3, and 7 days, and 2, 3, and 4 weeks posttransplantation, we calculated the mortality and assessed alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) levels in the serum of the model; evaluated the integrality and recovery of microcapsules; and stained with hematoxylin and eosin (H&E) the recovered microcapsules as well as the liver of the FHF rats., Results: Hepatocyte-specific functions, including the levels of ALT, AST, and ALB in the serum of the co-microencapsulation group, were significantly better than those in the other groups (p<0.05) from 2 to 4 weeks after transplantation. Moreover, cotransplantation of the microencapsulated hepatocytes and HUVECs decreased the mortality rate of the FHF rats. The recovered microcapsules were intact, and recovery was up to 90%. H&E staining showed that the microencapsulated cells were still alive, and the liver tissues had started to recover after 4 weeks posttransplantation., Conclusion: The microcapsules have good biocompatibility and immunoprotection to protect the hepatocytes from immunological rejection. Cotransplantation of the microencapsulated hepatocytes and HUVECs could decrease mortality rates and improve liver function in FHF.

Background: One of the major obstacles in regenerating thick, complex tissues such as the liver is their need for vascularization, which is essential to maintain cell viability during tissue growth and to induce structural organization. Herein, we have described a method to engineer a functional human vascular network., Methods: Enhanced green fluorescence protein-labeled human umbilical vein endothelial cells (GFP-HUVECs) were cocultivated with kusabira orange-labeled human mesenchymal stem cells (KO-hMSCs) inside a collagen/fibronectin matrix. Premature vascular network formation was visualized by fluorescence microscopy imaging. Furthermore, constructs prevascularized in vitro were implanted into a transparency window in immunodeficient mice., Results: Following several days of cultivation, GFP-HUVECs formed vessel-like structures that were stabilized by pericytes differentiated from KO-hMSCs. After implantation in vivo, the patency of human vascular structures was proved by rhodamine dextran infusion. These functional vascular structures remained for over 2 months., Discussion: Vascularization is the key challenge to organ generation. We successfully generated human vascular networks inside a matrix. Integration of parenchymal cells using our engineering technique should facilitate future efforts to reconstitute vascularized human organ systems in vitro., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: We quantified absolute myocardial blood flow (MBF) using a spin-labeling MRI (SL-MRI) method after transplantation of endothelial cells (ECs) into the infarcted heart. Our aims were to study the temporal changes in MBF in response to EC transplantation and to compare regional MBF with contractile function (wall motion) and microvascular density., Methods and Results: We first validated the SL-MRI method with the standard microsphere technique in normal rats. We then induced myocardial infarction in athymic rats and injected 5 million ECs (human umbilical vein endothelial cells) suspended in Matrigel or Matrigel alone (vehicle) along the border of the blanched infarcted area. At 2 weeks after myocardial infarction, MBF averaged over the entire slice (P=0.038) and in the infarcted region (P=0.0086) was significantly higher in EC versus vehicle group; the greater MBF was accompanied by an increase of microvasculature density in the infarcted region (P=0.0105 versus vehicle). At 4 weeks after myocardial infarction, MBF in the remote region was significantly elevated in EC-treated hearts (P=0.0277); this was accompanied by increased wall motion in this region assessed by circumferential strains (P=0.0075). Intraclass correlation coefficients and Bland-Altman plot revealed a good reproducibility of the SL-MRI method., Conclusions: MBF in free-breathing rats measured by SL-MRI is validated by the standard color microsphere technique. SL-MRI allows quantification of temporal changes of regional MBF in response to EC treatment. The proof-of-principle study indicates that MBF is a unique and sensitive index to evaluate EC-mediated therapy for the infarcted heart.

Cell transplantation is a promising strategy for therapeutic treatment of ischemic heart diseases. In this study, cord blood mesenchymal stem cells (cbMSCs) and human umbilical vein endothelial cells (HUVECs) in the form of core-shell bodies (cbMSC/HUVEC bodies) were prepared to promote vascularization and restore heart functions in an experimentally-created myocardial infarction (MI) rat model. Saline, cbMSC bodies and HUVEC bodies were used as controls. In vitro results indicated that cbMSC/HUVEC bodies possessed the capability of heterotypic assembly of cbMSCs and HUVECs into robust and durable tubular networks on Matrigel. The up-regulated gene expressions of VEGF and IGF-1 reflected the robust expansion of tubular networks; in addition, the augmented levels of SMA and SM22 suggested smooth muscle differentiation of cbMSCs, possibly helping to improve the durability of networks. Moreover, according to the in vivo echocardiographic, magnetic resonance and computed-tomographic results, transplantation of cbMSC/HUVEC bodies benefited post-MI dysfunction. Furthermore, the vascularization analyses demonstrated the robust vasculogenic potential of cbMSC/HUVEC bodies in vivo, thus contributing to the greater viable myocardium and the less scar region, and ultimately restoring the cardiac function. The concept of core-shell bodies composed of perivascular cells and endothelial cells may serve as an attractive cell delivery vehicle for vasculogenesis, thus improving the cardiac function significantly., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Previous studies have examined the therapeutic effect of endothelial progenitor cells (EPCs) during the chronic phase of cerebral infarction in rats; however, few studies have investigated the effects of EPCs during the acute phase of infarction. In this study, we evaluated the therapeutic effect of EPCs with low aldehyde dehydrogenase activity (Alde-Low EPCs) in rats with acute cerebral infarction, and our results provide insight that may help to identify a therapeutic mechanism of EPCs for acute cerebral infarction. The administration of Alde-Low EPCs into rats with acute cerebral infarction results in the accumulation and migration of the Alde-Low EPCs into the infarct area and the subsequent decrease of infarct volume. Moreover, we found that the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) signaling pathway may regulate the accumulation of Alde-Low EPCs. The transplantation of Alde-Low EPCs may represent a potential treatment strategy for acute cerebral infarction., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite the great potential of cell therapy for ischemic disease, poor cell survival after engraftment in ischemic tissue limits its efficacy. Here we tested a hypothesis that three-dimensionally grafted human umbilical vein endothelial cell (HUVEC) spheroids would exhibit improved angiogenic efficacy following transplantation into mouse ischemic limbs compared with HUVECs prepared by conventional two-dimensional monolayer culture. One day after surgical induction of hindlimb ischemia in athymic mice, HUVECs cultured in monolayer or HUVEC spheroids were transplanted intramuscularly into ischemic limbs. Four weeks after the treatment, in the spheroid HUVEC transplantation group, we observed increased hypoxia-inducible factor-1α expression, decreased apoptosis, and increased HUVEC survival in the ischemic tissue compared with the monolayer HUVEC transplantation group. Transplantation of HUVEC spheroids also resulted in enhanced and prolonged secretion of paracrine factors as well as enhanced expression of factors involved in the recruitment of circulating angiogenic progenitor cells. In summary, transplantation of HUVECs as spheroids enhanced cell survival, increased paracrine factor secretion, and showed a potential as a therapeutic method to treat ischemic tissue damages by promoting angiogenesis.

The therapeutic efficacy of cell-based therapy after stroke can be enhanced by making the host brain tissue more receptive to the administered cells, which thereby facilitates brain plasticity. We hypothesized that simvastatin increases human umbilical cord blood cell (HUCBC) migration into the ischemic brain and promotes brain plasticity and neurological functional outcome after stroke. Rats were subjected to 2-h middle cerebral artery occlusion (MCAo) and administered subtherapeutic doses of simvastatin (0.5 mg/kg, gavaged daily for 7 days), HUCBCs (1 × 10(6), one time injection via tail vein), or combination simvastatin with HUCBCs starting at 24 h after stroke. Combination treatment of stroke showed an interactive effect in improvement of neurological outcome compared with simvastatin or HUCBC monotherapy groups. In addition, combination treatment significantly increased brain-derived neurotrophic factor/TrkB expression and the number of engrafted HUCBCs in the ischemic brain compared with HUCBC monotherapy. The number of engrafted HUCBCs was significantly correlated with functional outcome (modified neurological severity score). Combination treatment significantly increased neurogenesis and synaptic plasticity in the ischemic brain, and promoted neuroblast migration in cultured subventricular zone explants. Using primary cultured neurons (PCNs), we found that combination treatment enhanced neurite outgrowth compared with nontreatment control, simvastatin or HUCBC supernatant monotherapy. Inhibition of TrkB significantly attenuated combination treatment-induced neurite outgrowth. Our data indicate that combination simvastatin and HUCBC treatment of stroke increases BDNF/TrkB expression, enhances HUCBC migration into the ischemic brain, amplifies endogenous neurogenesis, synaptic plasticity and axonal growth, and thereby improves functional outcome after stroke.

Objectives: During the last decade, the use of ex vivo-derived materials designed as implant scaffolds has increased significantly. This is particularly so in the area of regenerative medicine, or tissue engineering, where the natural chemical and biomechanical properties have been shown to be advantageous. By focusing on detailed events that occur during early-phase remodeling processes, our objective was to detail progressive changes in graft biomechanics to further our understanding of these processes., Methods: A perfusion bioreactor system and acellular human umbilical veins were used as a model three-dimensional vascular scaffold on which human myofibroblasts were seeded and cultured under static or defined pulsatile conditions. Cell function in relation to graft mechanical properties was assessed., Results: Cells doubled in density from approximately 1 × 10(6) to 2 ± 0.4 × 10(6) cells/cm ringlet, whereas static cultures remained unchanged. The material's compressive stiffness and ultimate tensile strength remained unchanged in both static and dynamic systems. However the Young's modulus values increased significantly in the physiologic range, whereas in the failure range, a significant reduction (66%) was shown under dynamic conditions., Conclusions: As pulse and flow conditions are modulated, complex mechanical changes are occurring that modify the elastic modulus differentially in both physiologic and failure ranges. Mechanical properties play an important role in graft patency, and a dynamic relationship between structure and function occurs during graft remodeling. These investigations have shown that as cells migrate into this ex vivo scaffold model, significant variation in material elasticity occurs that may have important implications in our understanding of early-stage vascular remodeling events., (Copyright © 2011 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Vaccination with xenogeneic and syngeneic endothelial cells is effective for inhibiting tumor growth. Nontoxic diphtheria toxin (CRM197), as an immunogen or as a specific inhibitor of heparin-binding EGF-like growth factor, has shown promising antitumor activity. Therefore, immunization with or administration of viable human umbilical vein endothelial cells (HUVECs) combined with CRM197 could have an enhanced antitumor effect. Six-week-old C57BL/6J male mice were vaccinated with viable HUVECs, 1 x 10(6) viable HUVECs combined with 100 μg CRM197, or 100 μg CRM197 alone by ip injections once a week for 4 consecutive weeks. RM-1 cells (5 x 10(5)) were inoculated by sc injection as a preventive procedure. During the therapeutic procedure, 6-week-old male C57BL/6J mice were challenged with 1 x 10(5) RM-1 cells, then injected sc with 1 x 10(6) viable HUVECs, 1 x 10(6) viable HUVECs + 100 μg CRM197, and 100 μg CRM197 alone twice a week for 4 consecutive weeks. Tumor volume and life span were monitored. We also investigated the effects of immunization with HUVECs on the aortic arch wall and on wound healing. Vaccination with or administration of viable HUVECs+CRM197 enhanced the inhibition of RM-1 prostatic carcinoma by 24 and 29%, respectively, and prolonged the life span for 3 and 4 days, respectively, compared with those of only vaccination or administration with viable HUVECs of tumor-bearing C57BL/6J mice. Furthermore, HUVEC immunization caused some damage to the aortic arch wall but did not have remarkable effects on the rate of wound healing; the wounds healed in approximately 13 days. Treatment with CRM197 in combination with viable HUVECs resulted in a marked enhancement of the antitumor effect in the preventive or therapeutic treatment for prostatic carcinoma in vivo, suggesting a novel combination for anti-cancer therapy.