Formation Of Functional Vessels Research Articles

DEVELOPING NEXT-GENERATION INJECTABLE ADIPOSE TISSUE GRAFTS FOR SOFT-TISSUE RECONSTRUCTION

IntroductionAutologous fat grafting has favourable potential as a regenerative strategy and is the current gold-standard to repair large contour defects, as needed in breast reconstruction after mastectomy and traumatic soft tissue reconstruction. Clinically, there is a limit on the volume of lipoaspirate which can be utilised to repair a soft-tissue defect. Surgical complications are the result of poor structural fidelity of lipoaspirate and graft resorption as a filling material and are hindered further by poor graft vascularisation. This study aims to develop injectable lipoaspirate-derived adipose tissue grafts with enhanced biologically and clinically-admissible structural and functional properties adopting light photocrosslinking of unmodified lipoaspirate.MethodsPatient-derived lipoaspirate was harvested and crosslinked using novel photoinitiator and exposure to visible light (wavelength 450nm) in surgery, establishing bonds between extracellular matrix (ECM) proteins within the material. The degree of crosslinking was tuned (photoinitiator concentration, light exposure, light intensity) and covalent bond formation measured using mass spectrometry. To predict patient response, SWATH-MS was used to identify differences in patient ECM and crosslinked grafts were implanted in vivo using a subcutaneous mouse model. Functional vessel formation and resorption were quantified using micro-CT and tissue-remodelling was assessed via histology.ResultsThere was an increase in the relative abundance of covalent bonds present with increasing degree of crosslinking. When injected, crosslinked lipoaspirate had better shape fidelity compared with native lipoaspirate – demonstrated by a smaller fibre diameter. Crosslinked lipoaspirate remained viable over long term culture and resulted in more predictable resorption profiles when implanted in vivo.ConclusionsThe crosslinking approach described here is tunable and functional across different patient samples. Improving the structural properties of lipoaspirate through minimal manipulation has clinical utility for the delivery of grafts with higher shape fidelity and therefore increased graft survival when implanted.

Nogo-B modulates physiological and pathological angiogenesis during development and disease

Angiogenesis, i.e., the formation of new blood vessels, is essential for normal development and functional vessel formation in ischemic diseases. During angiogenesis, endothelial cells (ECs) coordinate migration, proliferation, and metabolic fluxes to adapt to branching vessels' increasing energy demands. Nogo-B is a ubiquitously expressed reticulon family protein mainly localized to the endoplasmic reticulum (ER) membrane. Our previous studies have shown that the genetic loss of Nogo-B results in aberrant vascular remodeling and dysregulated endothelial barrier function. The current study aims to examine the role of endothelial Nogo-B in regulating developmental angiogenesis and pathological angiogenesis in retinopathy of prematurity (ROP). We hypothesized that endothelial Nogo-B is required for physiological and pathological angiogenesis, and Nogo-B depletion attenuates pathological angiogenesis. Using tamoxifen-inducible, endothelial cell-specific Nogo-B−/− (Nogo-BiECKO) mice, Nogo-B global knockout mice, and C57Bl/6 (WT) mice, our results show that loss of Nogo-B in ECs significantly delayed Isolectin B4 (IB4)-labeled retinal blood vessel sprouting. Impaired EC proliferation was observed by the staining of and incorporation of 5-ethynyl-2′-deoxyuridine (EdU). Furthermore, using the Oxygen Induced Retinopathy (OIR) model to mimic ROP, we could demonstrate that loss of Nogo-B in EC resulted in reduced neovascularization compared to WT. Mechanistically, using Percoll gradients, ultra-resolution microscopy, proximity ligation assay, and electron-microscopy, we show that Nogo-B is localized in the mitochondria-associated ER membrane (MAM) and regulated MAM remodeling. Using seahorse assay, quantitative RT-PCR, and metabolomics analysis, we determined that loss of Nogo-B altered VEGF-mediated EC glycolysis, which is needed for the energy demand during angiogenesis. These data suggest that Nogo-B regulates glucose metabolism in EC and plays a pivotal role in physiological and pathological angiogenesis. Targeting endothelial Nogo-B may provide a plausible approach to controlling the formation of pathological neovascularization in retinal diseases, such as ROP, diabetic retinopathy, and wet forms of age-related macular degeneration. This work was supported by the National Institutes of Health (R01HL153599) and awards from the American Heart Association award to JY. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiogenesis and Functional Vessel Formation Induced by Interstitial Flow and Vascular Endothelial Growth Factor Using a Microfluidic Chip

Angiogenesis occurs during both physiological and pathological processes. In this study, a microfluidic chip for the development of angiogenesis was utilized to assess angiogenic sprouting and functional vessel formation. We also found that vascular endothelial growth factor (VEGF) was a determinant of the initiation of vascular sprouts, while the direction of these sprouts was greatly influenced by interstitial flow. Isoforms of VEGF such as VEGF121, VEGF165, and VEGF189 displayed different angiogenic properties on the chip as assessed by sprout length and number, vessel perfusion, and connectivity. VEGF165 had the highest capacity to induce vascular sprouting among the three isoforms assessed and furthermore, also induced functional vessel formation. This chip could be used to analyze the effect of different angiogenic factors and drugs, as well as to explore the mechanism of angiogenesis induced by such factors.

Angiogenic Effects and Crosstalk of Adipose-Derived Mesenchymal Stem/Stromal Cells and Their Extracellular Vesicles with Endothelial Cells.

Adipose-derived mesenchymal stem/stromal cells (ASCs) are an adult stem cell population able to self-renew and differentiate into numerous cell lineages. ASCs provide a promising future for therapeutic angiogenesis due to their ability to promote blood vessel formation. Specifically, their ability to differentiate into endothelial cells (ECs) and pericyte-like cells and to secrete angiogenesis-promoting growth factors and extracellular vesicles (EVs) makes them an ideal option in cell therapy and in regenerative medicine in conditions including tissue ischemia. In recent angiogenesis research, ASCs have often been co-cultured with an endothelial cell (EC) type in order to form mature vessel-like networks in specific culture conditions. In this review, we introduce co-culture systems and co-transplantation studies between ASCs and ECs. In co-cultures, the cells communicate via direct cell–cell contact or via paracrine signaling. Most often, ASCs are found in the perivascular niche lining the vessels, where they stabilize the vascular structures and express common pericyte surface proteins. In co-cultures, ASCs modulate endothelial cells and induce angiogenesis by promoting tube formation, partly via secretion of EVs. In vivo co-transplantation of ASCs and ECs showed improved formation of functional vessels over a single cell type transplantation. Adipose tissue as a cell source for both mesenchymal stem cells and ECs for co-transplantation serves as a prominent option for therapeutic angiogenesis and blood perfusion in vivo.

Zebrafish Scube1 and Scube2 cooperate in promoting Vegfa signalling during embryonic vascularization.

The secreted and membrane-anchored signal peptide-CUB-EGF domain-containing proteins (SCUBE) gene family composed of three members was originally identified from endothelial cells (ECs). We recently showed that membrane SCUBE2 binds vascular endothelial growth factor (VEGF) and acts as a co-receptor for VEGF receptor 2 to modulate EC migration, proliferation, and tube formation during postnatal and tumour angiogenesis. However, whether these SCUBE genes cooperate in modulating VEGF signalling during embryonic vascular development remains unknown. To further dissect the genetic interactions of these scube genes, transcription activator-like effector nuclease-mediated genome editing was used to generate knockout (KO) alleles of each scube gene. No overt vascular phenotypes were seen in any single scube KO mutants because of compensation by other scube genes during zebrafish development. However, scube1 and scube2 double KO (DKO) severely impaired EC filopodia extensions, migration, and proliferation, thus disrupting proper vascular lumen formation during vasculogenesis and angiogenesis as well as development of the organ-specific intestinal vasculature. Further genetic, biochemical, and molecular analyses revealed that Scube1 and Scube2 might act cooperatively at the cell-surface receptor level to facilitate Vegfa signalling during zebrafish embryonic vascularization. We showed for the first time that cooperation between scube1 and scube2 is critical for proper regulation of angiogenic cell behaviours and formation of functional vessels during zebrafish embryonic development.

A model of guided cell self-organization for rapid and spontaneous formation of functional vessels.

Most achievements to engineer blood vessels are based on multiple-step manipulations such as manual sheet rolling or sequential cell seeding followed by scaffold degradation. Here, we propose a one-step strategy using a microfluidic coextrusion device to produce mature functional blood vessels. A hollow alginate hydrogel tube is internally coated with extracellular matrix to direct the self-assembly of a mixture of endothelial cells (ECs) and smooth muscle cells (SMCs). The resulting vascular structure has the correct configuration of lumen, an inner lining of ECs, and outer sheath of SMCs. These "vesseloids" reach homeostasis within a day and exhibit the following properties expected for functional vessels (i) quiescence, (ii) perfusability, and (iii) contractility in response to vasoconstrictor agents. Together, these findings provide an original and simple strategy to generate functional artificial vessels and pave the way for further developments in vascular graft and tissue engineering and for deciphering the angiogenesis process.

Cytokine-Like 1 Is a Novel Proangiogenic Factor Secreted by and Mediating Functions of Endothelial Progenitor Cells.

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. To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation. 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. 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.

Human Adipose Mesenchymal Stem Cells Show More Efficient Angiogenesis Promotion on Endothelial Colony-Forming Cells than Umbilical Cord and Endometrium.

Angiogenesis is a complicated process in which perivascular cells play important roles. Multipotent mesenchymal stem/stromal cells (MSCs) from distinct tissues have been proved to be proangiogenic and share functional properties and gene expression profiles with perivascular cells. However, different tissues derived MSCs may exhibit different potential for clinical applications. Accordingly, comparative studies on different MSCs are essential. Here, we characterized MSCs from adipose (ADSCs), umbilical cord (UCMSCs), and endometrium (EMSCs) in terms of the surface antigen expression, differentiation ability, and the ability of angiogenesis promotion on endothelial colony-forming cells (ECFCs) both in vitro and in vivo. No significant differences in immunophenotype and differentiation were observed. In addition, three types of MSCs all located around tubular-like structures formed by ECFCs in coculture system on matrigel. But ECFCs seeded on ADSCs monolayer formed more organized capillary-like network than that on UCMSCs or EMSCs. When suspended with ECFCs in matrigel and implanted into nude mice, ADSCs promoted more functional vessel formation after 7 days. Moreover, in murine hindlimb ischemia model, cotransplantation of ECFCs with ADSCs was significantly superior to UCMSCs and EMSCs in promoting perfusion recovery and limb salvage. Furthermore, ADSC-conditioned medium (CM) contained more proangiogenic factors (such as vascular endothelial growth factor-A, platelet-derived growth factor BB, and basic fibroblast growth factor) and less inhibitory factor (such as thrombospondin-1), when compared with UCMSC-CM and EMSC-CM. And ADSC-CM more durably stabilized the vascular-like structures formed by ECFCs on matrigel and promoted ECFCs migration more efficiently. In summary, MSCs from adipose show significantly efficient promotion on angiogenesis both in vitro and in vivo than UCMSCs and EMSCs. Hence, ADSCs may be recommended as a more suitable source for treating hindlimb ischemia.

Improved Left Ventricular Aneurysm Repair with Cell- and Cytokine-Seeded Collagen Patches.

Background Engineered heart tissues (EHTs) present a promising alternative to current materials for surgical ventricular restoration (SVR); however, the clinical application remains limited by inadequate vascularization postimplantation. Moreover, a suitable and economic animal model for primary screening is another important issue. Methods Recently, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride chemistry (EDC) to initiate a strengthened, cytokine-conjugated collagenous platform with a controlled degradation speed. In vitro, the biomaterial exhibited an enhanced mechanical strength maintaining a porous ultrastructure, and the constant release of cytokines promoted the proliferation of seeded human mesenchymal stem cells (hMSCs). In vivo, with the hMSC-seeded, cytokine-immobilized patch (MSCs + GF patch), we performed modified SVR for rats with left ventricular aneurysm postmyocardial infarction (MI). Overall, the rats that underwent modified SVR lost less blood and had lower mortality. After 4 weeks, the rats repaired with this cell-seeded, cytokine-immobilized patch presented preserved cardiac function, beneficial morphology, enhanced cell infiltration, and functional vessel formation compared with the cytokine-free (MSC patch), cell-free (GF patch), or blank controls (EDC patch). Furthermore, the degradable period of the collagen patch in vivo extended up to 3 months after EDC treatment. Conclusions EDC may substantially modify collagen scaffold and provide a promising and practical biomaterial for SVR.

Growth differentiation factor 15 promotes blood vessel growth by stimulating cell cycle progression in repair of critical-sized calvarial defect

Repair of large bone defects remains a challenge for surgeons, tissue engineering represents a promising approach. However, the use of this technique is limited by delayed vascularization in central regions of the scaffold. Growth differentiation factor 15(GDF15) has recently been reported to be a potential angiogenic cytokine and has an ability to promote the proliferation of human umbilical vein endothelial cells(HUVECs). Whether it can be applied for promoting vascularized bone regeneration is still unknown. In this study, we demonstrated that GDF15 augmented the expression of cyclins D1 and E, induced Rb phosphorylation and E2F-1 nuclear translocation, as well as increased HUVECs proliferation. Furthermore, we also observed that GDF15 promoted the formation of functional vessels at an artificially-induced angiogenic site, and remarkably improved the healing in the repair of critical-sized calvarial defects. Our results confirm the essential role of GDF15 in angiogenesis and suggest its potential beneficial use in regenerative medicine.

Proliferation status defines functional properties of endothelial cells.

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

The somite-secreted factor Maeg promotes zebrafish embryonic angiogenesis.

MAM and EGF containing gene (MAEG), also called Epidermal Growth Factor-like domain multiple 6 (EGFL6), belongs to the epidermal growth factor repeat superfamily. The role of Maeg in zebrafish angiogenesis remains unclear. It was demonstrated that maeg was dynamically expressed in zebrafish developing somite during a time window encompassing many key steps in embryonic angiogenesis. Maeg loss-of-function embryos showed reduced endothelial cell number and filopodia extensions of intersegmental vessels (ISVs). Maeg gain-of-function induced ectopic sprouting evolving into a hyperbranched and functional perfused vasculature. Mechanistically we demonstrate that Maeg promotes angiogenesis dependent on RGD domain and stimulates activation of Akt and Erk signaling in vivo. Loss of Maeg or Itgb1, augmented expression of Notch receptors, and inhibiting Notch signaling or Dll4 partially rescued angiogenic phenotypes suggesting that Notch acts downstream of Itgb1. We conclude that Maeg acts as a positive regulator of angiogenic cell behavior and formation of functional vessels.

EphrinB2/EphB4 pathway in postnatal angiogenesis: a potential therapeutic target for ischemic cardiovascular disease.

Ischemic cardiovascular disease remains one of the leading causes of morbidity and mortality in the world. Proangiogenic therapy appears to be a promising and feasible strategy for the patients with ischemic cardiovascular disease, but the results of preclinical and clinical trials are limited due to the complicated mechanisms of angiogenesis. Facilitating the formation of functional vessels is important in rescuing the ischemic cardiomyocytes. EphrinB2/EphB4, a novel pathway in angiogenesis, plays a critical role in both microvascular growth and neovascular maturation. Hence, investigating the mechanisms of EphrinB2/EphB4 pathway in angiogenesis may contribute to the development of novel therapeutics for ischemic cardiovascular disease. Previous reviews mainly focused on the role of EphrinB2/EphB4 pathway in embryo vascular development, but their role in postnatal angiogenesis in ischemic heart disease has not been fully illustrated. Here, we summarized the current knowledge of EphrinB2/EphB4 in angiogenesis and their interaction with other angiogenic pathways in ischemic cardiovascular disease.

Effects of dipeptidyl peptidase-4 (DPP-4) inhibition on angiogenesis and hypoxic injury in type 2 diabetes

AimsWe examined whether, in diabetic Ob/Ob mice, the dipeptidyl peptidase-4 (DPP-4) inhibitor (PKF275-055), an antihyperglycemic drug, that inhibits the biological inactivation of SDF-1 (stromal cell-derived factor-1), may increase endothelial progenitor cells (EPCs) mobilization and incorporation, which, in turn, may regenerate capillaries and reduce myocardial ischemia induced by strenuous exercise. Main methodsHalf of sixteen control and Ob/Ob mice and eight Ob/Ob mice treated with PKF275-055 for four weeks underwent a forced swim protocol. Oral glucose tolerance, circulating EPCs, capillary ultrastructure and density, hypoxic areas and SDF-1 localization in myocardium were measured. Key findingsOb/Ob mice were glucose intolerant, had a significant low number of circulating EPCs and myocardial capillaries compared to lean controls. The DPP-4 inhibitor significantly improved their glucose tolerance, doubled the number of circulating EPCs, stimulated the formation of functional vessels and SDF-1 localization in the endothelium of myocardial capillaries and arterioles. Cardiac hypoxia after forced swim in Ob/Ob mice was significantly reduced when they were treated with the DPP-4 inhibitor. SignificanceDPP-4 inhibition may re-establish an adequate capillary network in the myocardium of diabetic Ob/Ob mice by the mobilization and SDF-1-mediated incorporation of EPCs and, consequently, reducing the susceptibility to myocardial ischemic injury provoked by strenuous exercise.

Organization and function of the vascular network and its relation with satellite cells in normal and pathological muscle

During muscle regeneration, myogenic precursor cells (MPCs) interact with neighbouring cells, including endothelial cells (ECs). Previous studies showed that specific interactions take place between vessel cells and MPCs. However, little is known about their impact on MPC fate and the underlying molecular mechanisms in normal and pathological muscle. We demonstrated in vitro a functional interplay between ECs and MPCs as: – both cell type attract each other in migration assay, suggesting the secretion of specific attractive factors; – ECs strongly stimulate MPC differentiation; – MPCs promote angiogenesis, i.e. differentiation of ECs vessel-like structures. These results were confirmed in vivo, in which MPCs specifically promote the formation of functional vessels in a dose-dependent way. These results show that myogenesis and angiogenesis take place together. Several molecular candidates regulating angiogenesis/myogenesis coupling, including transcriptomic analysis of ECs and MPCs sorted at different time points during muscle regeneration, are under investigation in functional assays. Collectively our results show that specific interactions between MPCs and ECs couple myogenesis and angiogenesis during muscle regeneration. These interactions may be altered in degenerative myopathies, where we already demonstrated strong perturbations of the vascular network associated with functional alteration (weaker muscle perfusion).

2015 4thTERMIS World CongressBoston, MassachusettsSeptember 8–11, 2015

2015 4^thTERMIS World CongressBoston, MassachusettsSeptember 8–11, 2015

The Formin FMNL3 Controls Early Apical Specification in Endothelial Cells by Regulating the Polarized Trafficking of Podocalyxin

Angiogenesis is the fundamental process by which new blood vessels form from pre-existing vasculature. It plays a critical role in the formation of the vasculature during development and is triggered in response to tissue hypoxia in adult organisms. This process requires complex and coordinated regulation of the endothelial cell cytoskeleton to control cell shape and polarity. In our previous work, we showed that the cytoskeletal regulator FMNL3/FRL2 controls the alignment of stabilized microtubules during polarized endothelial cell elongation and that depletion of FMNL3 retards elongation of the intersegmental vessels in zebrafish. Recent work has shown that FMNL3 is also needed for vascular lumen formation, a critical element of the formation of functional vessels. Here, we show that FMNL3 interacts with Cdc42 and RhoJ, two Rho family GTPases known to be required for lumen formation. FMNL3 and RhoJ are concentrated at the early apical surface, or AMIS, and regulate the formation of radiating actin cables from this site. In diverse biological systems, formins mediate polarized trafficking through the generation of similar actin filaments tracks. We show that FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface--an important event in vascular lumenogenesis.

Adipose Stromal Cell Contact with Endothelial Cells Results in Loss of Complementary Vasculogenic Activity Mediated by Induction of Activin A.

Adipose stem/stromal cells (ASCs) after isolation produce numerous angiogenic growth factors. This justifies their use to promote angiogenesis per transplantation. In parallel, local coimplantation of ASC with endothelial cells (ECs) leading to formation of functional vessels by the donor cells suggests the existence of a mechanism responsible for fine-tuning ASC paracrine activity essential for vasculogenesis. As expected, conditioned media (CM) from ASC promoted ECs survival, proliferation, migration, and vasculogenesis. In contrast, media from EC-ASC cocultures had neutral effects upon EC responses. Media from cocultures exhibited lower levels of vascular endothelial growth factor (VEGF), hepatic growth factor, angiopoietin-1, and stromal cell-derived factor-1 compared with those in ASC CM. Activin A was induced in ASC in response to EC exposure and was responsible for overall antivasculogenic activity of EC-ASC CM. Except for VEGF, activin A diminished secretion of all tested factors by ASC. Activin A mediated induction of VEGF expression in ASC, but also upregulated expression of VEGF scavenger receptor FLT-1 in EC in EC-ASC cocultures. Blocking the FLT-1 expression in EC led to an increase in VEGF concentration in CM. In vitro pre-exposure of ASC to low number of EC before subcutaneous coimplantation with EC resulted in decrease in vessel density in the implants. In vitro tests suggested that activin A was partially responsible for this diminished ASC activity. This study shows that neovessel formation is associated with induction of activin A expression in ASC; this factor, by affecting the bioactivity of both ASC and EC, directs the crosstalk between these complementary cell types to establish stable vessels.

Total saponins from Albizia julibrissin inhibit vascular endothelial growth factor-mediated angiogenesis in vitro and in vivo.

Dried stem bark from Albizia julibrissin (AJ) is a highly valued Traditional Chinese Medicine, which has been shown to suppress tumor growth and angiogenesis. Total saponins from AJ (TSAJ) are one of the most bioactive components of AJ extract. The present study evaluated the anti-tumor and anti-angiogenic effects of TSAJ in vitro and in vivo. The anti-angiogenic activity of TSAJ was investigated by measuring the effects on vascular endothelial growth factor (VEGF)-induced proliferation, migration and tube formation of Ea.hy926 endothelial cells in vitro. The expression levels of proteins associated with VEGF-induced angiogenesis were determined by western blotting. Furthermore, in vivo Matrigel™ plug and H22 hepatoma tumor models were used to verify the anti-angiogenic effects of TSAJ. The present study demonstrated that TSAJ significantly inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation of Ea.hy926 cells in vitro. The anti-angiogenic effects of TSAJ were modulated by suppression of phosphorylated-(p-) focal adhesion kinase, p-Akt, and p-extracellular signal-regulated kinase in the VEGF/VEGF receptor 2 (R2) signaling pathway. Furthermore, oral administration of TSAJ significantly inhibited tumor growth and tumor-induced angiogenesis, as well as the formation of functional vessels, in the Matrigel™ plug model. These results suggest that TSAJ may be a potential anti-angiogenic agent that targets the VEGF/VEGFR2 signaling pathway, and inhibits tumor-induced angiogenesis.

Human Adipose Derived Mesenchymal Stromal Cells Show More Efficient Angiogeneic Promotion on EPCs Than Umbilical Cord and Endometrium

Angiogenesis is a complicated process involving endothelial proliferation, migration, and finally formation of functional vessels, in which perivascular cells play assistant roles. Mesenchymal stem cells (MSCs) from distinct human tissues have been proved to share functional properties and gene-expression profile with perivascular cells and to be proangiogenic. In this study we compared the proangiogenic ability of three types of MSC derived from human adipose stromal cells (ADSCs), umbilical cord (UCMSCs) and endometrium (EMSCs).Three types of MSCs all located around the tubular like structures formed by EPCs in coculture system on Matrigel in vitro (Figure 1A). However, different MSCs showed different potential for stimulating vessel formation of EPCs. After 6 days’ coculture, we found that EPCs seeded on ADSCs monolayer formed more organized capillary-like network (Figure 1B, P<0.01), and the length of vascular, number of junctions and ratio of vascular area was 18.96±3.52, 81.72±14.09 and 23.97%±2.11%, respectively (Figure 1C). EPCs seeded on UCMSCs only formed single tube-like structure (length, 2.90±1.31; junctions, 6.94±3.22; area, 3.05%±1.29%), while EPCs seeded on EMSCs gathered together not to form capillary-like structure (Figure 1B, C).Furthermore, we compared their proangiogenic potential of three types of MSCs. Matrigel contained MSCs and EPCs (ratio 3:2, total 2*106) was injected into nude mouse dorsal subcutaneous. 7 days following the injection, Matrigel was stripped off for the analysis of microvessels. As shown in Figure 2, three types of MSCs all promoted the formation of new microvessels. ADSC had the strongest stimulating effects, and formed more microvessels containing a large number of erythrocytes as shown by the appearance and HE staining (6.43%±2.57%). However, microvessel structures were not observed in the matrigel containing EPCs alone.Moreover, we assessed the therapeutic effects of co-transplantation of EPCs and MSCs in mouse hindlimb ischemic model. Right femoral artery ligation and excision was performed on anesthetized nude mice. After 24h of surgery, EPCs and MSCs were co-transplanted by tail intravenous injection (n=5, total 6.25*105; MSC/EPC ratio, 3:2). Blood flow of the ischemic limb was measured at 0, 7, 14, 21, 28 days after cell transplantation using Laser Doppler Perfusion Imager (Figure 3A). The results showed that the co-transplantation of EPCs with ADSCs significantly improved blood flow of ischemic limb after day 7 of transplantation (0.94±0.10, P<0.01, Figure 3B), and the final limb salvage rate in mice was significantly raised after day 28 of transplantation (Figure 3C, D). Thus, the blood stream reconstitute in early stage is the key factor for the final outcome of ischemic limb.We queried whether the different stimulation effects of MSCs on the vessel formation of EPCs are caused by MSC-secreted proangiogenic cytokines. Firstly, we analyzed the effects of MSC-CM on the proliferation and migration of EPCs in vitro, and found that migration rates/numbers of EPCs was highly increased by ADSC-CM (69.52%±9.66%, P<0.05) compared with UCMSC-CM and EMSC-CM. Next, we analyzed proangiogenic cytokines in MSC-CM using cytokine antibody array. The results showed that ADSCs secreted more proangiogenic cytokines such as VEGF-A, HGF, PDGF-BB and bFGF, and less inhibitor such as thrompospondin-1 in comparision to UCMSCs and EMSCs.In conclusion, MSCs could promote the formation of new microvessles of EPCs, and ADSCs are more efficient than UCMSCs or EMSCs by secreting more proangiogenic cytokines and less inhibitor. Thus ADSCs may be an ideal cellular source for the treatment of ischemia vascular disease. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.