Expression Of Flk-1 Research Articles

Reversine enhances the conversion of dedifferentiated fat cells into mature cardiomyocytes.

There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn't fully understood. We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. ANOVA with Tukey's post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p < 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p =0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p= 0 .001) increased significantly from day 7 until day 21. Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to cell stemness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Effects of different concentrations of reversine to enhance conversion of dedifferentiated fat cells into mature cardiomyocytes

Background There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn’t fully understood. Methods We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results ANOVA with Tukey’s post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p &lt; 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p =0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p= 0.001) increased significantly from day 7 until day 21. Conclusions Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to stem cell-ness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Flk1 Deficiency and Hypoxia Synergistically Promote Endothelial Dysfunction, Vascular Remodeling, and Pulmonary Hypertension.

The mechanisms underlying pulmonary hypertension (PH) remain largely unknown; further, why advanced vascular remodeling preferentially occurs in arterioles is yet to be answered. VEGF (vascular endothelial growth factor) regulates angiogenesis through Flk1 and Flt1 on endothelial cells (ECs), which may be related to PH pathogenesis. However, spatiotemporal expression patterns of Flk1 and Flt1 in the pulmonary vascular system and the role of endothelial Flk1 in PH development remain poorly understood. We analyzed multiple reporter mice, including Flk1-GFP bacterial artificial chromosome transgenic (Tg), Flt1-DsRed bacterial artificial chromosome Tg, and Flk1-GFP/Flt1-DsRed double Tg mice, to determine the spatiotemporal expression of Flk1 and Flt1 in hypoxia-induced PH. We also used Cdh5CreERT2/Flk1f/f/Tomato (Flk1-KO) mice to induce EC-specific Flk1 deletion and lineage tracing in chronic hypoxia. Flk1 was specifically expressed in the ECs of small pulmonary vessels, including arterioles. Conversely, Flt1 was more broadly expressed in the ECs of large- to small-sized vessels in adult mouse lungs. Intriguingly, Flk1+ ECs were transiently increased in hypoxia with proliferation, whereas Flt1 expression was unchanged. Flk1-KO mice did not exhibit pulmonary vascular remodeling nor PH in normoxia; however, the arteriolar ECs changed to a cuboidal shape with protrusion. In hypoxia, Flk1 deletion exacerbated EC dysfunction and reduced their number via apoptosis. Additionally, Flk1 deletion promoted medial thickening and neointimal formation in arterioles and worsened PH. Mechanistically, lineage tracing revealed that neointimal cells were derived from Flk1-KO ECs. Moreover, RNA sequencing in pulmonary ECs demonstrated that Flk1 deletion and hypoxia synergistically activated multiple pathways, including cell cycle, senescence/apoptosis, and cytokine/growth factor, concomitant with suppression of cell adhesion and angiogenesis, to promote vascular remodeling. Flk1 and Flt1 were differentially expressed in pulmonary ECs. Flk1 deficiency and hypoxia jointly dysregulated arteriolar ECs to promote vascular remodeling. Thus, dysfunction of Flk1+ ECs may contribute to the pathogenesis of advanced vascular remodeling in pulmonary arterioles.

A gene chip study suggests that miR-17-3p is associated with diabetic foot ulcers.

Background of the Study Diabetic foot ulcers (DFUs) are severe effect of diabetes. This research aimed to discover the role of micro-ribonucleic acid (miRNA) in treating DFUs involved in maggot debridement therapy (MDT) via a miRNA chip study. A miRNA chip approach was adopted. Patients with diabetes (type 1 or 2) who had at least one-foot ulcer (current or previous) were enrolled in the study. The alterations of miRNA expressions in the granulation tissue during treatment with MDT were measured. Following MDT, the increased expression of miR17-92 was verified in vivo. The miR-17-3p expression increased, and Flk-1 (vascular endothelial growth factor) expression was significantly reduced in patients with DFUs who received MDT (P < 0.01). Results from human umbilical vein endothelial cells that excrete or secrete showed consistency with in vitro findings (P< 0.001, P< 0.05). The overexpression of miR-17-3p demonstrated inhibitory activity on tube formation (P < 0.05). When DFUs were treated with MDT, it revealed that miR-17-3p had a negative regulatory effect on Flk-1.

Exosomes from human adipose-derived stem cells promoted the expression of angiogenic factors in endothelial cells

Introduction: In regenerative medicine, human adipose tissue–derived mesenchymal stem cells (hadMSCs) have exhibited tremendous promise as an efficient approach to chronic cardiovascular diseases. Noble effects of mesenchymal stem cells (MSCs) have been acknowledged to contribute to not only cellular engraftment and response to the site of injury but also paracrine action via the release of extracellular vesicles, notably exosomes. This study aimed to investigate the effects of exosomes from hadMSCs (hadMSC-Exo) on the angiogenesis of endothelial cells. Methods: Firstly, hadMSCs were characterized and cultured at a density of 500,000 cells in exosome-free medium for 48 hours. The conditioned medium was ultracentrifuged for exosome isolation. Obtained exosomes were characterized with the expression of CD9, CD63, and CD81 markers by flow cytometry and imaged with a TEM microscope. Exosomes were added to a culture medium of human umbilical vein endothelial cells (HUVECs) to understand the effects of exosomes on HUVECs. The expression of some angiogenic genes in HUVECs was identified by RT-qPCR. Results: The concentration of total protein in isolated exosomes by Bradford assay was 0.51 ± 0.04 mg/mL. Exosomes were successfully isolated and characterized by their morphology and immunophenotype. The results showed that exosomes significantly increased the mRNA expression of TGF (11.56 ± 2.03, P &lt; 0.05), Flk-1 (4.04 ± 0.01, P &lt; 0.05), VE-cadherin (7.25 ± 2.30, P &lt; 0.05), and CD31 (3.02 ± 1.13, P &lt; 0.05). Conclusion: This study suggested that exosomes promoted the angiogenesis of endothelial cells in vitro.

Effects of different concentrations of reversine to enhance conversion of dedifferentiated fat cells into mature cardiomyocytes

Background: There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn’t fully understood. Methods: We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results: ANOVA with Tukey’s post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p &lt; 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p = 0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p = 0.001) increased significantly from day 7 until day 21. Conclusions: Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to stem cell-ness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Endothelial GATA5 positively regulates angiogenesis via cathepsin S-mediated Angpt2/Flk1 and MMP2/9 signaling pathways

Although GATA5 is vital in maintaining the function of endothelial cells, the relationship between GATA5 and angiogenesis, however, remains unclear. Our study aims to determine how endothelial GATA5 mediates angiogenesis. Using the ischemic hindlimb of mice with GATA5 overexpression in the endothelia (EC-Ad mice), we showed that GATA5 overexpression could improve blood perfusion and increase capillary density. Furthermore, we showed that overexpression of GATA5 can increase the protein and mRNA levels of angiopoietin-2 (Angpt2) and fetal liver kinase 1 (Flk1) in the endothelia of EC-Ad mice, while GATA5 knockdown can inhibit the VEGF-165-induced proliferation, tube formation, and migration of human umbilical vein endothelial cells (HUVECs). In addition, we observed a decrease in the Angpt2 and Flk1, and the matrix metalloproteinase (MMP) family proteins: MMP2 and MMP9 while GATA5 was decreased. Meanwhile, our study also demonstrated that the expression of cathepsin S (Cat S) decreases when GATA5 is downregulated. Immunoprecipitation assay indicated that GATA5 could bind to Cat S directly. Furthermore, GATA5 or Cat S overexpression can promote tube formation and migration of HUVECs, restore the Angpt2 and Flk1 expression levels in the GATA5 knockdown HUVECs, and upregulate MMP2 and MMP9 protein levels. In summary, our study demonstrated that endothelial GATA5 could mediate angiogenesis by inducing the expression of Cat S, which mediates the Angpt2/Flk1 and MMP2/9 signaling pathways.

Possible testosterone redundancy for 5α-dihydrotestosterone in the masculinization of mouse external genitalia.

The development of embryonic external genitalia (eExG) into characteristic male structures, such as urethra and penile erectile tissues, depends on 5α-dihydrotestosterone (DHT). Although the corpus cavernosum (CC) is well known as essential for erectile function in adults, its developmental process and its dependency on DHT have been unknown. To reveal the dimorphic formation of the murine CC from the embryonic stage, we first analyzed the production of the protein vascular endothelial growth factor receptor-2 (FLK1) via its expression (hereinafter referred as "expression of FLK1") and the expression of alpha-smooth muscle actin (ACTA2) and collagen type 1 (COL1A1) in developing external genitalia. The 5-α reductase type 2 encoded by the SRD5A2 gene has been suggested to be a crucial enzyme for male sexual differentiation, as it converts testosterone (T) into DHT in the local urogenital organs. In fact, SRD5A2 mutation results in decreased synthesis of DHT, which leads to various degrees of masculinized human external genitalia (ExG). We further investigated the expression profile of SRD5A2 during the formation of the murine CC. We observed that SRD5A2 was expressed in smooth muscle of the CC. To determine the role of SRD5A2 in CC formation, we analyzed the formation of erectile tissue in the male Srd5a2 KO mice and measured the levels of androgens in the ExG by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Intriguingly, there were no obvious defects in the CCs of male Srd5a2 KO mice, possibly due to increased T levels. The current study suggests possible redundant functions of androgens in CC development.

Quantitative Analysis of Factors Regulating Angiogenesis for Stem Cell Therapy

Simple SummaryThe control of angiogenesis is essential in disease treatment or regenerative medicine. We conducted a clinical study of dedifferentiated fat (DFAT) cells, a kind of mesenchymal stem cells, by applying cell transplantation therapy to induce angiogenesis in patients with severe ischemic disease. This study aimed to analyze the effect of molecules that regulate angiogenesis in vitro and clarify their molecular mechanisms for therapeutic purposes. Normal human umbilical venous endothelial cells (HUVECs) were cultured in the presence of vascular endothelial growth factor (VEGF). Recombinant human angiopoietin-1-producing cells, conditioned media, mouse DFAT cells, and antioxidant polyphenols were added to this system at various concentrations. After 11 days, the cultures were immunostained with CD31 (PECAM-1), and microscopic images were subjected to analysis (area, length, joint, and path) by using software to quantitatively analyze blood vessel formation. The expression of angiogenic markers and COX pathway genes were analyzed by RT-PCR. As a result, the dose-dependent angiogenesis-promoting effect of rAng-1-producing cells, conditioned medium, or commercially available recombinant Ang-1 were observed. DFAT cells also promoted angiogenesis, whereas polyphenols inhibited angiogenesis in a dose-dependent manner.(1) Background: The control of angiogenesis is essential in disease treatment. We investigated angiogenesis-promoting or -suppressing factors and their molecular mechanisms. (2) Methods: Angiogenesis from HUVECs was quantitatively analyzed using the Angiogenesis Analysis Kit (Kurabo, Osaka, Japan). Human rAng-1-producing 107-35 CHO cells or mouse DFAT-D1 cells were co-cultured with HUVEC. Antioxidant polyphenols were added to the culture. Gene expression was analyzed by RT-PCR. (3) Results: The addition of rAng-1-producing cells, their culture supernatant, or commercially available rAng-1 showed a promoting effect on angiogenesis. The co-culture of DFAT-D1 cells promoted angiogenesis. Polyphenols showed a dose-dependent inhibitory effect on angiogenesis. Luteolin and quercetin showed remarkable anti-angiogenic effects. The expression of vWF, Flk1, and PECAM-1 was increased by adding rAng-1-producing cell culture supernatant. Polyphenols suppressed these genes. Apigenin and luteolin markedly suppressed α-SMA and Flk1. Resveratrol and quercetin enhanced the expression of PPARγ, and luteolin suppressed the expression of COX-1. The expression of endothelial nitric oxide synthase (eNOS), an oxidative stress-related gene, was slightly increased by luteolin. These results suggest that polyphenols induce ROS reduction. (4) Conclusions: We showed the promoting effect of Ang-1 or DFAT and the suppressing effect of polyphenols on angiogenesis and studied their molecular mechanisms. These results help control angiogenesis in regenerative therapy.

Expression profile of the Kisspeptin/Kiss1r system and angiogenic and immunological mediators in the ovary of cyclic and pregnant cats

The Kisspeptin/Kiss1r system has been studied in mammalian ovaries. However, there are still no studies on the modulation of this system and its relationship with angiogenic and immunological mediators in the ovary of domestic cats, especially during pregnancy. We evaluated the expression of Kisspeptin/Kiss1r and angiogenic and immunological mediators during folliculogenesis, luteogenesis and luteal regression of cyclic and pregnant cats. The ovary exhibited moderate to intense expression for Kiss1, VEGF, Flk-1, INFγ and MIF in oocytes and the follicular wall, while Kiss1r expression was low in granulosa cells. In these cells, there was also a greater expression of Kiss1, INFγ and MIF, mainly in secondary follicles, while tertiary and preovulatory follicles exhibited greater expression of VEGF and Flk-1 in this layer. In luteogenesis, Kiss1 immunostaining was higher in mature corpora lutea (MCL) of pregnant cats compared to vacuolated CL (VCL) and corpus albicans (CA). Pregnancy also increased the luteal gene expression of Kiss1 as well as Kiss1, Kiss1r, Flk-1, and MIF immunostaining in MCL, while reduced the area of VEGF expression in VCL and luteal mRNA expression of Mif when compared to non-pregnant animals. In addition, positive gene correlation between Kiss1r and Mif was observed in the CL. Kiss1, Kiss1r, Vegf and Mif expression were lower in the CA of cats in anestrus. These findings reveal that the expression of Kisspeptin/Kiss1r and angiogenic and immunological mediators, in the ovary of domestic cats, depend on the follicular and luteal stage, and the luteal expression of these mediators is influenced by pregnancy.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Kisspeptin/Kiss1r system and angiogenic and immunological mediators at the maternal-fetal interface of domestic cats.

The Kisspeptin/Kiss1r system is a key regulator of reproduction by stimulating gonadotrophin-releasing hormone and luteinizing hormone release, and in vitro studies have shown that Kisspeptin can modulate angiogenesis and immune function, factors that are also essential for reproduction However, there are no studies on the expression of Kisspeptin/Kiss1r at the maternal-fetal interface in domestic cats and its relationship with angiogenic and immunological mediators. Thus, our objective was to evaluate the spatiotemporal expression profile of Kisspeptin/Kiss1r and angiogenic and immunological mediators in the uterus and placenta of domestic cats during pregnancy. Uterus and placenta samples were collected from cats in mid pregnancy (N=6) and late pregnancy (N=6), in addition to uterus from non-pregnant cats in diestrus (N=7), to evaluate protein and gene expression of kisspeptin (Kiss1), kisspeptin receptor (Kiss1r), vascular endothelial growth factor (VEGF), tyrosine kinase receptor (Flk-1), placental growth factor (PLGF), interferon gamma (INFγ), migration inhibiting factor (MIF), tumor necrosis factor (TNFα), interleukins (IL6 and IL10) by immunohistochemistry and quantitative polymerase chain reaction. Pregnancy increased the uterine expression of Kiss1 and Kiss1r, especially at the late pregnancy, in addition to upregulating INFy, MIF, Vegf, Il10, and Tnf and downregulating Plgf. Higher placental expression of Kiss1r and Plgf mRNA occurred at the late pregnancy, while the expression of Kiss1, VEGF, Flk-1, INFy, TNFα, Il6, and IL10 was higher in the mid of pregnancy. A positive correlation between Kiss1 and Tnf was observed in the placenta, while Kiss1r had a negative correlation with Infγ, Il6, and Il10. The findings reveal that Kisspeptin/Kiss1r and angiogenic and immunological mediators at the maternal-fetal interface of pregnant cat have a gene correlation and are modulated by the gestational age. These data suggest possible functional links of Kisspeptin in placental angiogenesis and immunology.

The migratory pathways of the cells that form the endocardium, dorsal aortae, and head vasculature in the mouse embryo

BackgroundVasculogenesis in amniotes is often viewed as two spatially and temporally distinct processes, occurring in the yolk sac and in the embryo. However, the spatial origins of the cells that form the primary intra-embryonic vasculature remain uncertain. In particular, do they obtain their haemato-endothelial cell fate in situ, or do they migrate from elsewhere? Recently developed imaging techniques, together with new Tal1 and existing Flk1 reporter mouse lines, have allowed us to investigate this question directly, by visualising cell trajectories live and in three dimensions.ResultsWe describe the pathways that cells follow to form the primary embryonic circulatory system in the mouse embryo. In particular, we show that Tal1-positive cells migrate from within the yolk sac, at its distal border, to contribute to the endocardium, dorsal aortae and head vasculature. Other Tal1 positive cells, similarly activated within the yolk sac, contribute to the yolk sac vasculature. Using single-cell transcriptomics and our imaging, we identify VEGF and Apela as potential chemo-attractants that may regulate the migration into the embryo. The dorsal aortae and head vasculature are known sites of secondary haematopoiesis; given the common origins that we observe, we investigate whether this is also the case for the endocardium. We discover cells budding from the wall of the endocardium with high Tal1 expression and diminished Flk1 expression, indicative of an endothelial to haematopoietic transition.ConclusionsIn contrast to the view that the yolk sac and embryonic circulatory systems form by two separate processes, our results indicate that Tal1-positive cells from the yolk sac contribute to both vascular systems. It may be that initial Tal1 activation in these cells is through a common mechanism.

Adhesion of Flk1-expressing cells under shear flow in phospholipid polymer-coated immunoaffinity channels

Label-free cell separation is a promising method in the field of stem-cell research to obtain desired cell populations. Here, we report on phospholipid polymer-coated microfluidic channels with immobilized antibodies as devices for the capture of cells expressing target antigens in a label-free manner. We fabricated a microfluidic channel containing immobilized antibodies against vascular endothelial growth factor receptor 2 (Flk1), a potential marker for cardiac, angiogenic, and hematopoietic cell regeneration. A series of investigations was carried out to elucidate the effect of the immobilized antibodies on the adhesion behavior of the Flk1-expressing cell subpopulation derived from induced pluripotent stem cells. Increasing the immobilized antibody density (0.18–5.0 × 109 ligands mm−2) led to an increased number of cells adhering to the channel. The antibody-immobilized polymer-coated surface suppressed nonspecific cell adhesion, which was swept away by a weak shear flow, and captured Flk1-expressing cells under a wall shear stress of 1.7 Pa. Flk1 expression was 2.8-fold higher in the cells that adhered than in those that did not adhere. Therefore, an optimal antibody density and sweeping flow are required for effective label-free separation of Flk1-positive cells.

Cell Specific Expression of Vascular Endothelial Growth Factor Receptor-2 (Flk-1/KDR) in Developing Mice Embryo and Supporting Maternal Uterine Tissue during Early Gestation (D4-D7).

BackgroundVascular endothelial growth factor (VEGF) and the corresponding receptors play key role in vascu- logenesis and angiogenesis processes. VEGF is one of the prime candidates in regulating embryo implantation by increasing vascular permeability. VEGF receptor-2, also called Flk-1/KDR, is one of the prime receptor which is actively involved in the execution of various functions of VEGF. However, precise role of this receptor during early gestation period is yet to be addressed. In the present study, expression of Flk-1/KDR during peri-implantation mice uterus as well as fetal-maternal tissues from day 4-day 7 (D4-D7) of gestation was investigated.Materials and MethodsIn this experimental study, localization of Flk-1/KDR was investigated by immunohisto- chemistry and immunofluorescence techniques, in paraffin embedded tissue sections. Flk-1/KDR protein and mRNA expressions were investigated by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Effects of ovarian steroids on expression of Flk-1/KDR were also assessed by estrogen and progesterone antagonist treatment.ResultsUterine tissue on D4 showed strong expression of Flk-1/KDR in luminal and uterine glandular epithelium. On D5 and D6, differential expression of Flk-1/KDR was evidenced in certain cell types of the embryo, maternal tissues and fetal-maternal interface with varied intensity. Flk-1/KDR was specifically expressed in the ectoplacental cone (EPC) and various cells of the embryo on D7. Flk-1/KDR expression was not evidenced in the estradiol-17β (E2) and progesterone (P4) antagonist treated uterus. Western blotting result revealed presence of Flk-1/KDR protein in the all gestation days, except antagonist treated uterus. qRT-PCR analysis showed significant increase of Flk-1/KDR mRNA transcript on D6 and D7.ConclusionSpatial-temporal expression of Flk-1/KDR during peri-implntation period in mice uterus especially in the feto-maternal interface was observed. This spatio-temporal specificity as well as increased expression of Flk-1/KDR could be one of the determinants for establishment of fetal-maternal cross talk during the critical period of develop- ment.

5F peptide promotes endothelial differentiation of bone marrow stem cells through activation of ERK1/2 signaling

Synthetic apolipoprotein A-I (apoA-I) mimetic peptide 5F exhibits anti-atherosclerotic ability with largely unknown mechanism(s). Bone marrow (BM)-derived endothelial progenitor cells (EPCs) play a critical role in vascular integrity and function. The objective of the present study was to evaluate the effect of 5F on endothelial differentiation of BM stem cells and related mechanisms. Murine BM multipotent adult progenitor cells (MAPCs) were induced to differentiate into endothelial cells in vitro with or without 5F. The expression of endothelial markers vWF, Flk-1 and CD31 was significantly increased in the cells treated with 5F with enhanced in vitro vascular tube formation and LDL uptake without significant changes on proliferation and stem cell maker Oct-4 expression. Phosphorylated ERK1/2, not Akt, was significantly increased in 5F-treated cells. Treatment of MAPCs with PD98059 or small interfering RNA against ERK2 substantially attenuated ERK1/2 phosphorylation, and effectively prevented 5F-induced enhancement of endothelial differentiation of MAPCs. In vivo studies revealed that 5F increased EPCs number in the BM in mice after acute hindlimb ischemia that was effectively prevented with PD98059 treatment. These data supported the conclusion that 5F promoted endothelial differentiation of MAPCs through activation of ERK1/2 signaling.

1,25(OH)2D3 Strengthens the Vasculogenesis of Multipotent Mesenchymal Stromal Cells from Rat Bone Marrow by Regulating the PI3K/AKT Pathway.

BackgroundMultipotent mesenchymal stromal cells (MSCs) have recently been reported to promote vasculogenesis by differentiating into endothelial cells and releasing numerous cytokines and paracrine factors. However, due to low cell activity, their potential for clinical application is not very satisfactory. This study aimed to explore the effects and mechanisms of 1,25-dihydroxyvitamin D (1,25(OH)2D3) on the vasculogenesis of MSCs.MethodsMSCs were isolated from the femurs and tibias of rats and characterized by flow cytometry. After treatment with different concentrations of 1,25(OH)2D3 (0 µM, 0.1 µM and 1 µM), the proliferation of MSCs was analyzed by Cell Counting Kit-8 (CCK-8), and the migratory capability was measured by Transwell assays and cell scratch tests. Capillary-like structure formation was observed by using Matrigel. Western blotting was used to detect the expression of FLK-1 and vWF to investigate the differentiation of MSCs into endothelial cells. Western blotting and gelatin zymography were used to detect the expression and activities of VEGF, MMP-2 and MMP-9 secreted by MSCs under the influence of 1,25(OH)2D3. Finally, the VDR antagonist pyridoxal-5-phosphate (P5P) and the PI3K/AKT pathway inhibitor LY294002 were utilized to test the phosphorylation levels of key kinases in the PI3K/AKT pathway by Western blotting and the formation of capillary-like structures in Matrigel.ResultsThe proliferation and migratory capability of MSCs and the ability of MSCs to form a tube-like structure in Matrigel were enhanced after treatment with 1,25(OH)2D3. Moreover, MSCs treated with 1,25(OH)2D3 showed high expression of vWF and Flk-1. There was a significant increase in the expression of VEGF, MMP-2 and MMP-9 secreted by MSCs treated with 1,25(OH)2D3, as well as in the activity of MMP-2 and MMP-9. The phosphorylation level of AKT increased with time after 1,25(OH)2D3 treatment, while LY294002 weakened AKT phosphorylation. In addition, the ability to form capillary-like structures was reduced when the VDR and PI3K/AKT pathways were blocked.ConclusionThis study confirmed that 1,25(OH)2D3 treatment can strengthen the ability of MSCs to promote vasculogenesis in vitro, and the mechanism may be related to the activation of the PI3K/AKT pathway.

Expressions of vascular endothelial growth factor receptors, Flk1 and Flt1, in rat skin mast cells during development.

Vascular endothelial growth factor-A (VEGF-A) is a principal regulator of hematopoiesis as well as angiogenesis. However, the functions of VEGF-A and its receptors (VEGFRs) in the differentiation of mast cells (MCs) in the skin remain unclear. The aim of this study was to determine the expression patterns of two VEGFRs (Flk1 and Flt1) in the skin MCs during development and maturation in rats. From the 17th days of embryonic development (E17) to 1 day after birth (Day 1), most of skin MCs were immature cells containing predominant alcian blue (AB)+ rather than safranin O (SO)+ granules (AB>SO MCs). AB>SO MC proportions gradually decreased, while mature AB<SO MC proportions increased from Day 7 to 28. Flk1+ MC proportions increased from E20 and reached to approximately 90% from Day 1 to 21, thereafter decreased to about 10% at Day 60 and 90. Flk1+ MC proportions changed almost in parallel with the numbers of MCs and Ki67+ MC proportions from E17 to Day 90. The proportions of MCs with both nuclear and cytoplasmic Flt1-immunoreactivity were markedly increased at Day 28, when the proportions of nuclear Flk1+, Ki67+, and AB>SO MCs had significantly decreased, and AB<SO MC proportions significantly increased. Considering that the main function of Flt1 is suppression of Flk1 effects, our results indicated that cross-talk between Flk1 and Flt1 regulates the proliferation and maturation of the skin MCs during late embryonic and neonatal development in rats.

VEGF/Flk1 Mechanism is Involved in Roxarsone Promotion of Rat Endothelial Cell Growth and B16F10 Xenograft Tumor Angiogenesis

The potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1–50 μM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12–48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 μM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.

INFLAMMASOME-MEDIATED EXPANSION OF THE HEMATOPOIETIC SYSTEM IN THE VERTEBRATE EMBRYO

Embryonic hematopoietic stem and progenitor cells (HSPCs) have the capacity to rapidly expand while maintaining multilineage potential. As efforts to derive HSPCs in vitro are suboptimal, a better understanding of mechanisms regulating embryonic HSPC formation and expansion is needed. We previously reported that sterile inflammation stimulates embryonic HSPC production, yet it remains unclear how the embryo initiates inflammatory cues. The NLRP3 inflammasome cleaves and activates interleukin-1-beta (IL-1β) in response to sterile stimuli, including reactive oxygen species and metabolic flux. Building on previous findings that glucose exposure expands embryonic HSPCs, we hypothesized that these effects rely on inflammasome activation and IL-1β release. Glucose exposure promoted il1b expression; il1b knockdown suppressed the inductive effects of glucose on numbers of CD41+ HSPCs. Conversely, overexpression of the active form of il1b or pharmacologic inflammasome stimulation increased runx1/cmyb expression and numbers of Flk1+cMyb+ HSPCs. Inflammasome activation required NF-kB priming, and upregulated IL-1β targets IL-6 and IL-8, which are known to expand embryonic HSPCs. Loss of inflammasome components reduced HSPCs, and prevented their expansion in response to glucose stimulation. Furthermore, knockdown of pu.1/spi1l and cell ablation studies indicate that macrophages serve an important role in mediating the effects of inflammasome stimulation on HSPCs. Significantly, inflammasome stimulation of human iPSC-derived hemogenic precursors also increased the frequency of multilineage hematopoietic colony-forming units formed in culture. This work identifies the inflammasome as a conserved metabolic sensor that expands HSPC production in vivo and in vitro, which may ultimately promote human HSPC production for cell therapy.