Neovascularization by DPSC-ECs in a Tube Model for Pulp Regeneration Study.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.

Oxygen tension regulating hydrogels for vascularization and osteogenesis via sequential activation of HIF-1α and ERK1/2 signaling pathways in bone regeneration.

Angiogenesis plays a crucial role in bone regeneration. Hypoxia is a driving force of angiogenesis at the initial stage of tissue repair. The hypoxic microenvironment could activate the hypoxia-inducible factor (HIF)-1α signaling pathway in cells, thereby enhancing the proliferation, migration and pro-angiogenic functions of stem cells. However, long-term chronic hypoxia could inhibit osteogenic differentiation and even lead to apoptosis. Therefore, shutdown of the HIF-1α signaling pathway and providing oxygen at later stage probably facilitate osteogenic differentiation and bone regeneration. Herein, an oxygen tension regulating hydrogel that sequentially activate and deactivate the HIF-1α signaling pathway were prepared in this study. Its effect and mechanism on stem cell differentiation were investigated both in vitro and in vivo. We proposed a gelatin-based hydrogel capable of sequentially delivering a hypoxic inducer (copper ions) and oxygen generator (calcium peroxide). The copper ions released from the hydrogels significantly enhanced cell viability and VEGF secretion of BMSCs via upregulating HIF-1α expression and facilitating its translocation into the nucleus. Additionally, calcium peroxide promoted alkaline phosphatase activity, osteopontin secretion, and calcium deposition through the activation of ERK1/2. Both Cu2+ and calcium peroxide demonstrated osteogenic promotion individually, while their synergistic effect within the hydrogels led to a superior osteogenic effect by potentially activating the HIF-1α and ERK1/2 signaling pathways.

Ultrasonographic analysis of palatal donor site healing accelerated with platelet-rich fibrin following subepithelial connective tissue harvesting.

OBJECTIVE: Platelet-rich fibrin (PRF) contains a variety of growth factors and bioactive molecules that play crucial roles in wound healing and angiogenesis. We aimed to evaluate the effects of PRF on tissue thickness and vascularization of the palatal donor site by ultrasound (USG) following subepithelial connective tissue harvesting. METHODOLOGY: A subepithelial connective tissue graft was harvested from the palatal region with a single incision for root coverage in 20 systemically healthy patients. In the test group (n = 10), the PRF membrane was placed at the donor site, whereas no material was applied in the control group (n=10). Palatal tissue thickness (PTT) and pulsatility index (PI) were evaluated by USG at baseline and on the 3rd, 7th, 14th, 30th, and 90th days after surgery. The early healing index (EHI) was used to evaluate donor site healing for 30 days. RESULTS: PTT was significantly higher in the PRF group on the 3rd and 14th days after surgery when compared to the controls. In the PRF-treated group, PI levels were significantly higher than in the controls, especially on the 14th day. PTT increased significantly 90 days after surgery compared to the test group baseline, but controls showed a significant decrease. The PRF group showed statistically significant improvements in EHI scores compared to controls on days 3, 7, and 14. This study found a negative correlation between PI values and EHI scores on postoperative days three and seven in the test group. CONCLUSION: USG is a non-invasive, objective method to radiographically evaluate the regenerative effects of PRF on palatal wound healing after soft tissue harvesting. To overcome graft inadequacy in reharvesting procedures, PRF application may enhance clinical success and reduce possible complications by increasing tissue thickness and revascularization in the donor area.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

A Novel Mechanism of MSCs Responding to Occlusal Force for Bone Homeostasis.

Alveolar bone, as tooth-supporting bone for mastication, is sensitive to occlusal force. However, the mechanism of alveolar bone loss after losing occlusal force remains unclear. Here, we performed single-cell RNA sequencing of nonhematopoietic (CD45-) cells in mouse alveolar bone after removing the occlusal force. Mesenchymal stromal cells (MSCs) and endothelial cell (EC) subsets were significantly decreased in frequency, as confirmed by immunofluorescence and flow cytometry. The osteogenic and proangiogenic abilities of MSCs were impaired, and the expression of mechanotransducers yes associated protein 1 (Yap) and WW domain containing transcription regulator 1 (Taz) in MSCs decreased. Conditional deletion of Yap and Taz from LepR+ cells, which are enriched in MSCs that are important for adult bone homeostasis, significantly decreased alveolar bone mass and resisted any further changes in bone mass induced by occlusal force changes. Interestingly, LepR-Cre; Yapf/f; Tazf/f mice showed a decrease in CD31hi endomucin (Emcn)hi endothelium, and the expression of some EC-derived signals acting on osteoblastic cells was inhibited in alveolar bone. Mechanistically, conditional deletion of Yap and Taz in LepR+ cells inhibited the secretion of pleiotrophin (Ptn), which impaired the proangiogenic capacity of LepR+ cells. Knockdown in MSC-derived Ptn repressed human umbilical vein EC tube formation in vitro. More important, administration of recombinant PTN locally recovered the frequency of CD31hiEmcnhi endothelium and rescued the low bone mass phenotype of LepR-Cre; Yapf/f; Tazf/f mice. Taken together, these findings suggest that occlusal force governs MSC-regulated endothelium to maintain alveolar bone homeostasis through the Yap/Taz/Ptn axis, providing a reference for further understanding of the relationship between dysfunction and bone homeostasis.

Enhancing bone tissue engineering using iron nanoparticles and magnetic fields: A focus on cytomechanics and angiogenesis in the chicken egg chorioallantoic membrane model.

AIM: To evaluate the potential of iron nanoparticles (FeNPs) in conjunction with magnetic fields (MFs) to enhance osteoblast cytomechanics, promote cell homing, bone development activity, and antibacterial capabilities, and to assess their in vivo angiogenic viability using the chicken egg chorioallantoic membrane (CAM) model. SETTINGS AND DESIGN: Experimental study conducted in a laboratory setting to investigate the effects of FeNPs and MFs on osteoblast cells and angiogenesis using a custom titanium (Ti) substrate coated with FeNPs. MATERIALS AND METHODS: A custom titanium (Ti) was coated with FeNPs. Evaluations were conducted to analyze the antibacterial properties, cell adhesion, durability, physical characteristics, and nanoparticle absorption associated with FeNPs. Cell physical characteristics were assessed using protein markers, and microscopy, CAM model, was used to quantify blood vessel formation and morphology to assess the FeNP-coated Ti's angiogenic potential. This in vivo study provided critical insights into tissue response and regenerative properties for biomedical applications. STATISTICAL ANALYSIS: Statistical analysis was performed using appropriate tests to compare experimental groups and controls. Significance was determined at P < 0.05. RESULTS: FeNPs and MFs notably improved osteoblast cell mechanical properties facilitated the growth and formation of new blood vessels and bone tissue and promoted cell migration to targeted sites. In the group treated with FeNPs and exposed to MFs, there was a significant increase in vessel percentage area (76.03%) compared to control groups (58.11%), along with enhanced mineralization and robust antibacterial effects (P < 0.05). CONCLUSION: The study highlights the promising potential of FeNPs in fostering the growth of new blood vessels, promoting the formation of bone tissue, and facilitating targeted cell migration. These findings underscore the importance of further investigating the mechanical traits of FeNPs, as they could significantly advance the development of effective bone tissue engineering techniques, ultimately enhancing clinical outcomes in the field.

Matrix Resistance Toward Proteolytic Cleavage Controls Contractility-Dependent Migration Modes During Angiogenic Sprouting.

Tissue homeostasis and disease states rely on the formation of new blood vessels through angiogenic sprouting, which is tightly regulated by the properties of the surrounding extracellular matrix. While physical cues, such as matrix stiffness or degradability, have evolved as major regulators of cell function in tissue microenvironments, it remains unknown whether and how physical cues regulate endothelial cell migration during angiogenesis. To investigate this, a biomimetic model of angiogenic sprouting inside a tunable synthetic hydrogel is created. It is shown that endothelial cells sense the resistance of the surrounding matrix toward proteolytic cleavage and respond by adjusting their migration phenotype. The resistance cells encounter is impacted by the number of covalent matrix crosslinks, crosslink degradability, and the proteolytic activity of cells. When matrix resistance is high, cells switch from a collective to an actomyosin contractility-dependent single cellular migration mode. This switch in collectivity is accompanied by a major reorganization of the actin cytoskeleton, where stress fibers are no longer visible, and F-actin aggregates in large punctate clusters. Matrix resistance is identified as a previously unknown regulator of angiogenic sprouting and, thus, provides a mechanism by which the physical properties of the matrix impact cell migration modes through cytoskeletal remodeling.

Clay Sculpture-Inspired 3D Printed Microcage Module Using Bioadhesion Assembly for Specific-Shaped Tissue Vascularization and Regeneration.

3D bioprinting techniques have enabled the fabrication of irregular large-sized tissue engineering scaffolds. However, complicated customized designs increase the medical burden. Meanwhile, the integrated printing process hinders the cellular uniform distribution and local angiogenesis. A novel approach is introduced to the construction of sizable tissue engineering grafts by employing hydrogel 3D printing for modular bioadhesion assembly, and a poly (ethylene glycol) diacrylate (PEGDA)-gelatin-dopamine (PGD) hydrogel, photosensitive and adhesive, enabling fine microcage module fabrication via DLP 3D printing is developed. The PGD hydrogel printed micocages are flexible, allowing various shapes and cell/tissue fillings for repairing diverse irregular tissue defects. In vivo experiments demonstrate robust vascularization and superior graft survival in nude mice. This assembly strategy based on scalable 3D printed hydrogel microcage module could simplify the construction of tissue with large volume and complex components, offering promise for diverse large tissue defect repairs.

Framework Nucleic Acids-Based VEGF Signaling Activating System for Angiogenesis: A Dual Stimulation Strategy.

Angiogenesis is crucial for tissue engineering, wound healing, and regenerative medicine. Nanomaterials constructed based on specific goals can be employed to activate endogenous growth factor-related signaling. In this study, based on the conventional single-stranded DNA self-assembly into tetrahedral framework nucleic acids (tFNAs), the Apt02 nucleic acid aptamer and dimethyloxallyl glycine (DMOG) small molecule are integrated into a complex via a template-based click chemistry reaction and toehold-mediated strand displacement reaction. Thus, being able to simulate the VEGF (vascular endothelial growth factor) function and stabilize HIF (hypoxia-inducible factor), a functional whole is constructed and applied to angiogenesis. Cellular studies demonstrate that the tFNAs-Apt02 complex (TAC) has a conspicuous affinity to human umbilical vein endothelial cells (HUVECs). Further incubation with DMOG yields the tFNAs-Apt02-DMOG complex (TACD), which promotes VEGF secretion, in vitro blood vessel formation, sprouting, and migration of HUVECs. Additionally, TACD enhances angiogenesis by upregulating the VEGF/VEGFR and HIF signaling pathways. Moreover, in a diabetic mouse skin defect repair process, TACD increases blood vessel formation and collagen deposition, therefore accelerating wound healing. The novel strategy simulating VEGF and stabilizing HIF promotes blood-vessel formation in vivo and in vitro and has the potential for broad applications in the vascularization field.

The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Human lens epithelial-secreted exosomes attenuate ocular angiogenesis via inhibiting microglial activation.

The lens is an avascular tissue, where epithelial cells (LECs) are the primary living cells. The role of LECs-derived exosomes (LEC-exos) is largely unknown. In our study, we determined the anti-angiogenic role of LEC-exos, manifested as regressed retinal neovascularization (NV) using the oxygen-induced retinopathy (OIR), and reduced choroidal NV size and pathological vascular leakage using the laser-induced choroidal neovascularization (laser-induced CNV). Furthermore, the activation and accumulation of microglia were also restricted by LEC-exos. Based on Luminex multiplex assays, the expressions of chemokines such as SCYB16/CXCL16, MCP-1/CCL2, I-TAC/CXCL11, and MIP 3beta/CCL19 were decreased after treatment with LEC-exos. Transwell assays showed that LEC-exos restricted the migration of the mouse microglia cell line (BV2 cells). After incubation with LEC-exos-treated BV2 cells, human umbilical vein endothelial cells (hUVECs) were collected for further evaluation using tube formation, Transwell assays, and 5-ethynyl-2'-deoxyuridine (EDU) assays. Using in vitro experiments, the pro-angiogenic effect of microglia was restricted by LEC-exos. Hence, it was investigated that LEC-exos attenuated ocular NV, which might attribute to the inhibition of microglial activation and accumulation.

Osteopontin Promotes Angiogenesis in the Spinal Cord and Exerts a Protective Role Against Motor Function Impairment and Neuropathic Pain After Spinal Cord Injury.

STUDY DESIGN: Basic science study using a hemisection spinal cord injury (SCI) model. OBJECTIVE: We sought to assess the effect of blocking osteopontin (OPN) upregulation on motor function recovery and pain behavior after SCI and to further investigate the possible downstream target of OPN in the injured spinal cord. SUMMARY OF BACKGROUND DATA: OPN is a noncollagenous extracellular matrix protein widely expressed across different tissues. Its expression substantially increases following SCI. A previous study suggested that this protein might contribute to locomotor function recovery after SCI. However, its neuroprotective potential was not fully explored, nor were the underlying mechanisms. MATERIALS AND METHODS: We constructed a SCI mouse model and analyzed the expression of OPN at different time points and the particular cell distribution in the injured spinal cord. Then, we blocked OPN upregulation with lentivirus-delivering siRNA targeting OPN specifically and examined its effect on motor function impairment and neuropathic pain after SCI. The underlying mechanisms were explored in the OPN-knockdown mice model and cultured vascular endothelial cells. RESULTS: The proteome study revealed that OPN was the most dramatically increased protein following SCI. OPN in the spinal cord was significantly increased three weeks after SCI. Suppressing OPN upregulation through siRNA exacerbated motor function impairment and neuropathic pain. In addition, SCI resulted in an increase in vascular endothelial growth factor (VEGF), AKT phosphorylation, and angiogenesis within the spinal cord, all of which were curbed by OPN reduction. Similarly, OPN knockdown suppressed VEGF expression, AKT phosphorylation, cell migration, invasion, and angiogenesis in cultured vascular endothelial cells. CONCLUSION: OPN demonstrates a protective influence against motor function impairment and neuropathic pain following SCI. This phenomenon may result from the proangiogenetic effect of OPN, possibly due to activation of the VEGF and/or AKT pathways.

Composite Microparticles of Fat Graft and GFR Matrigel Improved Volume Retention by Promoting Cell Migration and Vessel Regeneration.

BACKGROUND: The retention volume of autologous fat grafts decreases after transplantation due to limited nutrition infiltration and insufficient blood supply. Structural fat grafts and the 3M (multipoint, multitunnel, and multilayer) injection technique have been considered to improve the survival of grafts; however, it is difficult for surgeons to practice in the clinic because grafts tend to gather into a cluster, especially in large volume fat grafting. Therefore, we hypothesize that prefabricated microparticle fat grafts (PFMG) may improve the retention rate. METHODS: The C57BL/6 mouse fat particles were embedded in growth factor-reduced (GFR)-Matrigel to detect cell migration by immunofluorescence staining in vitro. PFMG was prepared by mixing mouse fat particles and GFR Matrigel in a 1:1 volume ratio and injected subcutaneously into C57BL/6 mice. Fat particles mixed with PBS in equal volume served as control group. The grafts were harvested at 1, 4, 8, and 12 weeks after sacrifice. The retention rate of grafts at each time point was measured, and the structural alterations were detected by SEM. Fat necrosis and blood vessel density were evaluated by histological analysis. RESULTS: CD34+ cells are migrated from the PFMG and formed a tree-like tubular network in the in vitro study. The retention rate was higher in the PFMG group than in the control group at week 12 (38% vs. 30%, p < 0.05). After transplantation, the dissociated structure of fat particles was maintained in PFMG by SEM analysis. Histological analysis of PFMG confirmed less fat necrosis and more blood vessel density in the PFMG group at the early stage than in the control group. The GFR Matrigel was displaced by adipose tissue with time. CONCLUSIONS: In this study, we developed a novel fat grafting method, PFMG that dispersed fat grafts and maintained the structure after transplantation. High volume retention volume of PFMG was achieved by promoting cell migration and vessel regeneration. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Three dimensional (bio)printing of blood vessels: from vascularized tissues to functional arteries.

Tissue engineering has emerged as a strategy for producing functional tissues and organs to treat diseases and injuries. Many chronic conditions directly or indirectly affect normal blood vessel functioning, necessary for material exchange and transport through the body and within tissue-engineered constructs. The interest in vascular tissue engineering is due to two reasons: (1) functional grafts can be used to replace diseased blood vessels, and (2) engineering effective vasculature within other engineered tissues enables connection with the host's circulatory system, supporting their survival. Among various practices, (bio)printing has emerged as a powerful tool to engineer biomimetic constructs. This has been made possible with precise control of cell deposition and matrix environment along with the advancements in biomaterials. (Bio)printing has been used for both engineering stand-alone vascular grafts as well as vasculature within engineered tissues for regenerative applications. In this review article, we discuss various conditions associated with blood vessels, the need for artificial blood vessels, the anatomy and physiology of different blood vessels, available 3D (bio)printing techniques to fabricate tissue-engineered vascular grafts and vasculature in scaffolds, and the comparison among the different techniques. We conclude our review with a brief discussion about future opportunities in the area of blood vessel tissue engineering.

GroEL of Porphyromonas gingivalis-induced microRNAs accelerate tumor neovascularization by downregulating thrombomodulin expression in endothelial progenitor cells.

We found that GroEL in Porphyromonas gingivalis accelerated tumor growth and increased mortality in tumor-bearing mice; GroEL promoted proangiogenic function, which may be the reason for promoting tumor growth. To understand the regulatory mechanisms by which GroEL increases the proangiogenic function of endothelial progenitor cells (EPCs), we explored in this study. In EPCs, MTT assay, wound-healing assay, and tube formation assay were performed to analyze its activity. Western blot and immunoprecipitation were used to study the protein expression along with next-generation sequencing for miRNA expression. Finally, a murine tumorigenesis animal model was used to confirm the results of in vitro. The results indicated that thrombomodulin (TM) direct interacts with PI3 K/Akt to inhibit the activation of signaling pathways. When the expression of TM is decreased by GroEL stimulation, molecules in the PI3 K/Akt signaling axis are released and activated, resulting in increased migration and tube formation of EPCs. In addition, GroEL inhibits TM mRNA expression by activating miR-1248, miR-1291, and miR-5701. Losing the functions of miR-1248, miR-1291, and miR-5701 can effectively alleviate the GroEL-induced decrease in TM protein levels and inhibit the proangiogenic abilities of EPCs. These results were also confirmed in animal experiments. In conclusion, the intracellular domain of the TM of EPCs plays a negative regulatory role in the proangiogenic capabilities of EPCs, mainly through direct interaction between TM and PI3 K/Akt to inhibit the activation of signaling pathways. The effects of GroEL on tumor growth can be reduced by inhibiting the proangiogenic properties of EPCs through the inhibition of the expression of specific miRNAs.

Computational modeling of angiogenesis: The importance of cell rearrangements during vascular growth.

Angiogenesis is the process wherein endothelial cells (ECs) form sprouts that elongate from the pre-existing vasculature to create new vascular networks. In addition to its essential role in normal development, angiogenesis plays a vital role in pathologies such as cancer, diabetes and atherosclerosis. Mathematical and computational modeling has contributed to unraveling its complexity. Many existing theoretical models of angiogenic sprouting are based on the "snail-trail" hypothesis. This framework assumes that leading ECs positioned at sprout tips migrate toward low-oxygen regions while other ECs in the sprout passively follow the leaders' trails and proliferate to maintain sprout integrity. However, experimental results indicate that, contrary to the snail-trail assumption, ECs exchange positions within developing vessels, and the elongation of sprouts is primarily driven by directed migration of ECs. The functional role of cell rearrangements remains unclear. This review of the theoretical modeling of angiogenesis is the first to focus on the phenomenon of cell mixing during early sprouting. We start by describing the biological processes that occur during early angiogenesis, such as phenotype specification, cell rearrangements and cell interactions with the microenvironment. Next, we provide an overview of various theoretical approaches that have been employed to model angiogenesis, with particular emphasis on recent in silico models that account for the phenomenon of cell mixing. Finally, we discuss when cell mixing should be incorporated into theoretical models and what essential modeling components such models should include in order to investigate its functional role. This article is categorized under: Cardiovascular Diseases > Computational Models Cancer > Computational Models.

Transluminal Pillars-Their Origin and Role in the Remodelling of the Zebrafish Caudal Vein Plexus.

Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been described in developing vasculature of many organs and organisms. The aim of this study was to resolve the question about pillar formation and their further maturation employing zebrafish caudal vein plexus (CVP). The CVP development was monitored by in vivo confocal microscopy in high spatio-temporal resolution using the transgenic zebrafish model Fli1a:eGPF//Gata1:dsRed. We tracked back the formation of pillars (diameter ≤ 4 µm) and intercapillary meshes (diameter > 4 µm) and analysed their morphology and behaviour. Transluminal pillars in the CVP arose via a combination of sprouting, lumen expansion, and/or the creation of intraluminal folds, and those mechanisms were not associated directly with blood flow. The follow-up of pillars indicated that one-third of them disappeared between 28 and 48 h post fertilisation (hpf), and of the remaining ones, only 1/17 changed their cross-section area by >50%. The majority of the bigger meshes (39/62) increased their cross-section area by >50%. Plexus simplification and the establishment of hierarchy were dominated by the dynamics of intercapillary meshes, which formed mainly via sprouting angiogenesis. These meshes were observed to grow, reshape, and merge with each other. Our observations suggested an alternative view on intussusceptive angiogenesis in the CVP.

Hydrogels with tunable mechanical plasticity regulate endothelial cell outgrowth in vasculogenesis and angiogenesis.

The endothelial cell (EC) outgrowth in both vasculogenesis and angiogenesis starts with remodeling surrounding matrix and proceeds with the crosstalk between cells for the multicellular vasculature formation. The mechanical plasticity of matrix, defined as the ability to permanently deform by external traction, is pivotal in modulating cell behaviors. Nevertheless, the implications of matrix plasticity on cell-to-cell interactions during EC outgrowth, along with the molecular pathways involved, remain elusive. Here we develop a collagen-hyaluronic acid based hydrogel platform with tunable plasticity by using compositing strategy of dynamic and covalent networks. We show that although the increasing plasticity of the hydrogel facilitates the matrix remodeling by ECs, the largest tubular lumens and the longest invading distance unexpectedly appear in hydrogels with medium plasticity instead of the highest ones. We unravel that the high plasticity of the hydrogels promotes stable integrin cluster of ECs and recruitment of focal adhesion kinase with an overenhanced contractility which downregulates the vascular endothelial cadherin expression and destabilizes the adherens junctions between individual ECs. Our results, further validated with mathematical simulations and in vivo angiogenic tests, demonstrate that a balance of matrix plasticity facilitates both cell-matrix binding and cell-to-cell adherens, for promoting vascular assembly and invasion.

Adipose-Derived Mesenchymal Stem Cells Protect Endothelial Cells from Hypoxic Injury by Suppressing Terminal UPR In Vivo and In Vitro.

Adipose-derived stem cells (ASCs) have been used as a therapeutic intervention for peripheral artery disease (PAD) in clinical trials. To further explore the therapeutic mechanism of these mesenchymal multipotent stromal/stem cells in PAD, this study was designed to test the effect of xenogeneic ASCs extracted from human adipose tissue on hypoxic endothelial cells (ECs) and terminal unfolded protein response (UPR) in vitro and in an atherosclerosis-prone apolipoprotein E-deficient mice (ApoE-/- mice) hindlimb ischemia model in vivo. ASCs were added to Cobalt (II) chloride-treated ECs; then, metabolic activity, cell migration, and tube formation were evaluated. Fluorescence-based sensors were used to assess dynamic changes in Ca2+ levels in the cytosolic- and endoplasmic reticulum (ER) as well as changes in reactive oxygen species. Western blotting was used to observe the UPR pathway. To simulate an acute-on-chronic model of PAD, ApoE-/- mice were subjected to a double ligation of the femoral artery (DLFA). An assessment of functional recovery after DFLA was conducted, as well as histology of gastrocnemius. Hypoxia caused ER stress in ECs, but ASCs reduced it, thereby promoting cell survival. Treatment with ASCs ameliorated the effects of ischemia on muscle tissue in the ApoE-/- mice hindlimb ischemia model. Animals showed less muscle necrosis, less inflammation, and lower levels of muscle enzymes after ASC injection. In vitro and in vivo results revealed that all ER stress sensors (BIP, ATF6, CHOP, and XBP1) were activated. We also observed that the expression of these proteins was reduced in the ASCs treatment group. ASCs effectively alleviated endothelial dysfunction under hypoxic conditions by strengthening ATF6 and initiating a transcriptional program to restore ER homeostasis. In general, our data suggest that ASCs may be a meaningful treatment option for patients with PAD who do not have traditional revascularization options.

Enhanced pericyte-endothelial interactions through NO-boosted extracellular vesicles drive revascularization in a mouse model of ischemic injury.

Despite improvements in medical and surgical therapies, a significant portion of patients with critical limb ischemia (CLI) are considered as "no option" for revascularization. In this work, a nitric oxide (NO)-boosted and activated nanovesicle regeneration kit (n-BANK) is constructed by decorating stem cell-derived nanoscale extracellular vesicles with NO nanocages. Our results demonstrate that n-BANKs could store NO in endothelial cells for subsequent release upon pericyte recruitment for CLI revascularization. Notably, n-BANKs enable endothelial cells to trigger eNOS activation and form tube-like structures. Subsequently, eNOS-derived NO robustly recruits pericytes to invest nascent endothelial cell tubes, giving rise to mature blood vessels. Consequently, n-BANKs confer complete revascularization in female mice following CLI, and thereby achieve limb preservation and restore the motor function. In light of n-BANK evoking pericyte-endothelial interactions to create functional vascular networks, it features promising therapeutic potential in revascularization to reduce CLI-related amputations, which potentially impact regeneration medicine.