Early Blood Vessel Research Articles

Vitamin D3 Release from MgO Doped 3D Printed TCP Scaffolds for Bone Regeneration.

Regenerating bone tissue in critical-sized craniofacial bone defects remains challenging and requires the implementation of innovative bone implants with early stage osteogenesis and blood vessel formation. Vitamin D3 is incorporated into MgO-doped 3D-printed scaffolds for defect-specific and patient-specific implants in low load-bearing areas. This novel bone implant also promotes early stage osteogenesis and blood vessel development. Our results show that vitamin D3-loaded MgO-doped 3D-printed scaffolds enhance osteoblast cell proliferation 1.3-fold after being cultured for 7 days. Coculture studies on osteoblasts derived from human mesenchymal stem cells (hMSCs) and osteoclasts derived from monocytes show the upregulation of genes related to osteoblastogenesis and the downregulation of RANK-L, which is essential for osteoclastogenesis. Release of vitamin D3 also inhibits osteoclast differentiation by 1.9-fold after a 21-day culture. After 6 weeks, vitamin D3 release from MgO-doped 3D-printed scaffolds enhances the new bone formation, mineralization, and angiogenic potential. The multifunctional 3D-printed scaffolds can improve early stage osteogenesis and blood vessel formation in craniofacial bone defects.

Optical imaging technology realizes early tumor diagnosis by detecting angiogenesis.

The occurrence and development of blood vessels play a key role in different stages of tumor growth, while current imaging techniques are difficult to detect early tumor angiogenesis because of their low sensitivity. Therefore, this article introduces high-sensitivity optical imaging technology to achieve early tumor diagnosis by detecting tumor angiogenesis. Liver and pancreatic tumor models in nude mice were respectively established to represent tumors with a rich or poor blood supply. The two optical imaging methods, in vivo confocal fluorescence imaging and photoacoustic imaging, were used to detect tumor angiogenesis at different stages. Finally, the changes in blood vessels were verified by immunostaining. Both autoluminescence imaging and pathological staining confirmed that these two tumor models were successfully established. In vivo confocal fluorescence imaging found that the early tumor blood vessel structure had obvious characteristics: disorder, tortuous deformation, thin diameter, which were significantly different from the normal tissues. Photoacoustic imaging could effectively identify blood vessels inside early tumors, which were small and disordered and might be used as one of the predictors of early tumor development. CD31 immunostaining was used to evaluate the vascular status of tumors at different stages and under different blood supply conditions. The vascular structures observed under the microscope in the two tumor models were consistent with the results observed by optical imaging methods. The optical imaging methods could monitor the characteristics of angiogenesis in the rich or poor blood supply tumors, especially the early diagnosis of tumors.

Fabrication of succinate-alginate xerogel films for in vitro coupling of osteogenesis and neovascularization

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 μM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 μM.

Rab35 governs apicobasal polarity through regulation of actin dynamics during sprouting angiogenesis

In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35’s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.

EHD2 modulates Dll4 endocytosis during blood vessel development.

ObjectiveDespite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta‐like ligand 4 (Dll4) endocytosis during Notch activation.Approach and ResultsUsing both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane‐bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE‐cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling.ConclusionsOverall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation.

Polymer Composite Sponges with Inherent Antibacterial, Hemostatic, Inflammation-Modulating and Proregenerative Performances for Methicillin-Resistant Staphylococcus aureus-Infected Wound Healing.

Clinical wound management remains a major challenge due to massive bleeding, bacterial infection, and difficult wound healing after tissue trauma. To simultaneously address these issues, composite polymer sponges for accelerating drug-resistant bacterial infected wound healing are fabricated by facilely mixing sodium polyacrylate (PAAS), double quaternary ammonium salts-conjugated chitosan (QAS-CS), and collagen (COL) in aqueous solution, followed by lyophilization. Composite sponges (PAAS/QAS-CS/COL, PQC) show highly porous microstructures (porosity ≈90%) with moderate compress modulus (≈0.3 MPa), tensile strength (0.004 MPa), and high swelling ratio (≈3500%). Importantly, PQC sponge demonstrates superior hemostasis ability over commercially available CS sponge by inducing rapid hemagglutination, and exhibits significantly better antibacterial activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli by destroying cell membrane and causing the leakage of bioactive components such as potassium ion and β-galactosidase from treated bacterial. Furthermore, PQC sponge can efficiently promote cell proliferation. Significantly, the sponge greatly expedites the regeneration of MRSA-infected full-thickness skin wound in rabbit by successfully eradicating bacterial infection, and reducing inflammation. PQC sponge also improves both early angiogenesis and blood vessel maturation at the wound site. Overall, this multifunctional sponge is a promising wound dressing for clinical use and holds great potential for rapid clinical translation.

Osteogenesis and angiogenesis of a bulk metallic glass for biomedical implants

Implantation is an essential issue in orthopedic surgery. Bulk metallic glasses (BMGs), as a kind of novel materials, attract lots of attentions in biological field owing to their comprehensive excellent properties. Here, we show that a Zr61Ti2Cu25Al12 (at. %) BMG (Zr-based BMG) displays the best cytocompatibility, pronounced positive effects on cellular migration, and tube formation from in-vitro tests as compared to those of commercial-pure titanium and poly-ether-ether-ketone. The in-vivo micro-CT and histological evaluation demonstrate the Zr-based BMG can significantly promote a bone formation. Immunofluorescence tests and digital reconstructed radiographs manifest a stimulated effect on early blood vessel formation from the Zr-based BMG. Accordingly, the intimate connection and coupling effect between angiogenesis and osteogenesis must be effective during bone regeneration after implanting Zr-based BMG. Dynamic gait analysis in rats after implanting Zr-based BMG demonstrates a tendency to decrease the pain level during recovery, simultaneously, without abnormal ionic accumulation and inflammatory reactions. Considering suitable mechanical properties, we provide a realistic candidate of the Zr61Ti2Cu25Al12 BMG for biomedical applications.

Thermal injury induces early blood vessel occlusion in a porcine model of brass comb burn

Burn wound progression is an important determinant of patient morbidity and mortality after injury. In this study, we used the brass comb contact burn to determine burn wound vertical injury progression with a focus on blood vessel occlusion and endothelial cell death. Class A 3-month-old Yorkshire pigs received a brass comb contact burn. Burn wounds were sampled at 0, 30 min, 1, 2, 4, and 24 h. Hematoxylin Phloxin Saffron staining and vimentin immunostaining were performed to determine the depth of blood vessel occlusion and endothelial cell death, respectively. The depth of blood vessel occlusion increased by 30 min (p < 0.005) and peaked by 1 to 4 h (p > 0.05). The depth of endothelial cell death risen to a plateau at 30 min (p < 0.005) to 2 h and then peaked at 24 h (p < 0.03). We observed a progression of blood vessel occlusion and vascular endothelial cell death from the middle of the dermis to the hypodermis within 2 h to 4 h after the initial injury, namely a progression from a second-degree (partial thickness) to third-degree (full thickness) burn. These data suggest that therapeutic interventions during this time window may provide a better outcome by reducing or preventing vertical progression of blood vascular occlusion or endothelial cell death.

Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

Single Cell Transcriptome Data Analysis Defines the Heterogeneity of Peripheral Nerve Cells in Homeostasis and Regeneration.

The advances in single-cell RNA sequencing technologies and the development of bioinformatics pipelines enable us to more accurately define the heterogeneity of cell types in a selected tissue. In this report, we re-analyzed recently published single-cell RNA sequencing data sets and provide a rationale to redefine the heterogeneity of cells in both intact and injured mouse peripheral nerves. Our analysis showed that, in both intact and injured peripheral nerves, cells could be functionally classified into four categories: Schwann cells, nerve fibroblasts, immune cells, and cells associated with blood vessels. Nerve fibroblasts could be sub-clustered into epineurial, perineurial, and endoneurial fibroblasts. Identified immune cell clusters include macrophages, mast cells, natural killer cells, T and B lymphocytes as well as an unreported cluster of neutrophils. Cells associated with blood vessels include endothelial cells, vascular smooth muscle cells, and pericytes. We show that endothelial cells in the intact mouse sciatic nerve have three sub-types: epineurial, endoneurial, and lymphatic endothelial cells. Analysis of cell type-specific gene changes revealed that Schwann cells and endoneurial fibroblasts are the two most important cell types promoting peripheral nerve regeneration. Analysis of communication between these cells identified potential signals for early blood vessel regeneration, neutrophil recruitment of macrophages, and macrophages activating Schwann cells. Through this analysis, we also report appropriate marker genes for future single cell transcriptome data analysis to identify cell types in intact and injured peripheral nerves. The findings from our analysis could facilitate a better understanding of cell biology of peripheral nerves in homeostasis, regeneration, and disease.

A Novel Acellular Dermal Scaffold Prepared Using High-Intensity Focused Ultrasound Energy for the Repair of Soft Tissue Defects

Abstract Objective To evaluate a naturally derived acellular dermal scaffold for soft tissue reconstruction using high-intensity focused ultrasound energy (HIFU). Materials and Methods Acellular dermal scaffolds (ADSs) were prepared by purification of bovine skin. Half of the scaffolds were subjected to high-intensity focused ultrasound energy (HIFU) to modify collagen structure, whereas the other half was used as control. A large skin defect was made in the dorsum of white mice, and the scaffolds were used to cover the induced defects. Wound healing was evaluated histologically after 2, 6, and 12 weeks using common and specific stained sections (n = 20). Statistical Analysis Mean values and standard deviations were calculated for each group, and Student’s t-test was used for statistical analysis (α= 0.05; n = 20). Results After 2 weeks, all examined specimens revealed the presence of inflammatory cellular infiltration and early immature blood vessel formation. After 6 weeks, inflammatory cellular infiltration was reduced, with evidence of maturation of new blood vessels observed for all groups. After 12 weeks, there was a significant increase (F = 124, p &lt; 0.01) in new collagen formation and count of mature blood vessels observed for the HIFU group compared with control. Evidence of remodeling of new collagen fibers and biodegradation of the grafts was also observed. Conclusions HIFU-modified ADSs enhanced wound healing and could be used to cover large soft tissue defects.

Observation of the early blood vessels of cutaneous malignant melanoma using Swept Source Optical Coherence Tomography Angiography (SS-OCTA)

Melanoma, characterized by high mortality, rapid development and accompanied with angiogenesis is the most typical malignant tumor in skin cancer. Hence, the detection of blood vessels is of much significance. The early vascular network has small scale. If we remove the tumor early and biopsy it, it will increase the spread of the cancer cells and infection and bleeding. In this case, we presented a new angiography method. A high-resolution OCT system for noninvasive angiographic imaging of early skin melanoma — Swept Source Optical Coherence Tomography Angiography (SS-OCTA) is proposed. With a high lateral resolution of 10[Formula: see text][Formula: see text]m in vivo tomographic angiography, SS-OCTA is used to image and identify the morphology of the early tumor blood vessels. In addition, a control group experiment is conducted to observe the growth of melanoma in the process of rupture, malformation of micro-vessels. The results of the analysis and statistical test ([Formula: see text]) are statistically significant.

PREVALENCE OF METABOLIC SYNDROME IN PATIENTS WITH ARTERIAL HYPERTENSION AND ITS IMPACT ON ASYMPTOMATIC CAROTID ATHEROSCLEROSIS

The importance of metabolic syndrome (MetSy) lies in the fact that its components are proven risk factors for early blood vessel atherosclerosis and thrombosis.Aim of the paper: Our aim was to establish the prevalence of MetSy in patients with arterial hypertension (AH) and its impact on asymptomatic carotid atherosclerosis. The study involved 391 examinees, divided into two groups. The study group consisted of patients with arterial hypertension (n=342; average age, 66.56 ± 09.52; with 51% of female gender). The presence of cardiovascular (CV) risk factors was established for all involved patients, cardiovascular risk score was determined (SCORE risk), laboratory analyses were performed, as well as anthropometric measurements and color Doppler sonography of the great blood vessels of the neck. The patients with AH were divided into two groups according to the presence of MetSy. Metabolic syndrome was confirmed in 198 patients who comprised group I; there were 144 examinees without MetSy and these comprised group II. Those with MetSy had a greater average number of CV risk factors, a higher SCORE risk score, higher body mass index (p&lt;0.0001), and more frequently had diabetes, hyperlipidemia and obesity. The thickness of the intimal medial complex (IMC) of the carotid arteries was significantly greater in the group with MetSy (p&lt;0.0001) – 51% of examinees had IMC thickness ≥0.90. The patients with MetSy more commonly had one or more carotid plaques (p=0.03), a higher average number of plaques (p=0.01) and percentage of stenosis (p=0.01). As the most important factors associated with early carotid atherosclerosis, multivariant regression analysis singled out the following (for the model R=0.512. R2=0.262. adjusted R2=0.255. standard error of the estimate = 0.174; p&lt;0.0001): age (coefficiant β=0.331. p&lt;0.0001), number of MetSy components (coefficient β=0.158. p=0.002), level of serum uric acid (coefficient β=0.284; p&lt;0.0001). Our results demonstrated a significant association of MetSy and its components with early atherosclerotic changes in the carotid arteries.

The protection effect of ethyl pyruvate in early nerve blood vessels after traumatic brain injury

The protection effect of ethyl pyruvate in early nerve blood vessels after traumatic brain injury

Adipose-Derived Stem Cell Condition Medium Enhances Expression of Ephrin-B2 Related to Neovascularization in Burn Wound Tissue

The transmembrane ligand ephrin-B2 is molecular marker for arterial endothelial cells and early blood vessel formation. Neovascularization is one of the important event that support wound healing. Adiposederived stem cells conditioned medium (ADSC-CM) is known to contain cytokines and growth factors as regenerative agent. This study aims to investigate the effect of ADSC-CM on ephrin-B2 expression that represent as neovascularization marker in burn wound granulation tissue. Male Sprague dawley rats were randomly divided into control, conditioned medium (CM) and medium complete (MC) groups. Full-thickness burn wounds were created by placing preheated metal plat on dorsal thorax of the prepared rats. The burn wounds were treated once daily according to their group. Skin wound tissues were collected at day 7, 14, and 21 post wounded for histological analysis. The expression of ephrin-B2 was demonstrated by immunohistochemistry staining. Neovascularization evaluation was done by calculating the number of newly formed capillaries that expressed Ephrin-B2 in wound granulation tissues. Expression of Ephrin-B2 in burn wound tissue was prominently increased in CM compared to control and MC groups on day 7, 14 and 21 post wounded. The CM group also showed higher neovascular number in granulation tissue compared to control and MC group on day 7, 14 and 21 (p &lt; 0.05). Our results showed prominent ephrin-B2 expression in burn wound tissue after treated with ADSC-CM. Topical application of ADSC-CM has shown to promote angiogenesis in full-thickness burn wound in part through the increased of ephrin-B2 expression. Therefore ADSC-CM could act as an alternative strategy to promote burn wound healing.

A Snake Venom-Secreted Phospholipase A2 Induces Foam Cell Formation Depending on the Activation of Factors Involved in Lipid Homeostasis.

MT-III, a snake venom GIIA sPLA2, which shares structural and functional features with mammalian GIIA sPLA2s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (MΦs) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA2 are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-γ and PPAR-β/δ and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-γ, PPAR-β/δ, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-β/δ, but not PPAR-γ, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-β/δ and PPAR-γ upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.

BRG1 (Brahma-Related Gene 1) Promotes Endothelial Mrtf Transcription to Establish Embryonic Capillary Integrity

The chromatin remodeling enzyme BRG1 (brahma-related gene 1) transcriptionally regulates target genes important for early blood vessel development and primitive hematopoiesis. However, because Brg1 deletion in vascular progenitor cells results in lethal anemia by embryonic day 10.5 (E10.5), roles for BRG1 in embryonic vascular development after midgestation are unknown. In this study, we sought to determine whether endothelial cell BRG1 regulates genes important for vascular development or maintenance later in embryonic development. Using mice with temporally inducible deletion of endothelial BRG1 (Brg1fl/fl;Cdh5(PAC)-CreERT2 ), we found that Brg1 excision between E9.5 and 11.5 results in capillary dilation and lethal hemorrhage by E14.5. This phenotype strongly resembles that seen when the SRF (serum response factor) transcription factor is deleted from embryonic endothelial cells. Although expression of Srf and several of its known endothelial cell target genes are downregulated in BRG1-depleted endothelial cells, we did not detect binding of BRG1 at these gene promoters, indicating that they are not direct BRG1 target genes. Instead, we found that BRG1 binds to the promoters of the SRF cofactors Mrtfa and Mrtfb (myocardin-related transcription factors A and B) in endothelial cells, and these genes are downregulated in Brg1-deficient endothelial cells. BRG1 promotes transcription of endothelial Mrtfa and Mrtfb, which elevates expression of SRF and SRF target genes that establish embryonic capillary integrity. These data highlight a new and temporally specific role for BRG1 in embryonic vasculature and provide novel information about epigenetic regulation of Mrtf expression and SRF signaling in developing blood vessels.

Enrichment of Scleroderma Vascular Disease-Associated Autoantigens in Endothelial Lineage Cells.

Scleroderma patients with autoantibodies to CENPs and/or interferon-inducible protein 16 (IFI-16) are at increased risk of severe vascular complications. This study was undertaken to determine whether these autoantigens are enriched in cells of the vasculature. Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI-16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI-16 and CD31 expression were defined in paraffin-embedded skin sections from scleroderma patients and from healthy controls. IFI-16 expression was determined by flow cytometric analysis in circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells. Expression of CENP-A, IFI-16, and CD31 was enriched in EBs on days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI-16, CD31, and CENPs A and B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of paraffin-embedded skin sections showed enrichment of IFI-16 in CD31-positive vascular endothelial cells in biopsy specimens from scleroderma patients and normal controls. Flow cytometric analysis revealed IFI-16 expression in circulating hematopoietic progenitor cells but minimal expression in CECs. Our findings indicate that expression of the scleroderma autoantigens IFI-16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens.

Detection of initial angiogenesis from dorsal aorta into metanephroi and elucidation of its role in kidney development.

Reconstruction of blood vessels is considered the most difficult part for the complicated organs, therefore, blood vessel construction is regarded as a key point for kidney regeneration in vitro. Vasculogenesis and angiogenesis are the two mechanisms to form blood vessels in embryonic organs, and most studies resided in vaculogenesis. Angiogenesis resided mostly in adult diseases such as wound healing, growth of tumors, and psoriasis diseases. However, renal angiogenesis is simply attributed to the sprouting of pre-existing blood vessel from dorsal aorta into metanephroi, and its occurrence is considered to be at a late stage of metanephric development. Since no techniques are available for delicate detection, the initial angiogenesis from dorsal aorta into metanephroi as well as its role in kidney development still remained unclear. In this study, we developed a method to detect the initial angiogenesis of dorsal aorta into metanephroi, and firstly clarified that dorsal aorta angiogenesis occurred at an early stage of metanephric development. We also elucidated the role of dorsal aorta angiogenesis in promoting the early blood vessel formation, tubule formation and glomeruli maturation. It is suggested that blood flow and dynamic circulation of various factors at the early developing stage may be prerequisite to a successful construction of blood vessels in the complicated organs either in vitro or in vivo. These findings contribute to a better understanding of dorsal aorta angiogenesis during kidney development and shed light on its significant value for the application of tissue engineering to complicated organs.

Annexin A3 Regulates Early Blood Vessel Formation.

Annexins are a large family of calcium binding proteins that associate with cell membrane phospholipids and are involved in various cellular processes including endocytosis, exocytosis and membrane-cytoskeletal organization. Despite studies on numerous Annexin proteins, the function of Annexin A3 (Anxa3) is largely unknown. Our studies identify Anxa3 as a unique marker of the endothelial and myeloid cell lineages of Xenopus laevis during development. Anxa3 transcripts are also detected in endothelial cells (ECs) of zebrafish and mouse embryos, suggesting an important evolutionary function during formation of blood vessels. Indeed, Anxa3 loss-of-function experiments in frog embryos reveal its critical role during the morphogenesis of early blood vessels, as angioblasts in MO injected embryos fail to form vascular cords. Furthermore, in vitro experiments in mammalian cells identify a role for Anxa3 in EC migration. Our results are the first to reveal an in vivo function for Anxa3 during vascular development and represent a previously unexplored aspect of annexin biology.