VEGF Induction Research Articles

Arctigenin Prevents Retinal Edema in a Murine Retinal Vein Occlusion Model.

Macular edema causes vision loss in patients with retinal vein occlusion (RVO) and diabetic macular edema (DME). The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is used for treatment; however, this therapy is invasive, and recurrence occurs in some cases. The establishment of a non-invasive treatment would help to solve these problems. Here, we focused on arctigenin, a lignan polyphenol found in burdock sprout, and has effects on inflammatory and microcirculatory when taken orally. We hypothesized that oral intake of arctigenin could be effective against retinal edema in RVO and DME. In this study, the degree of retinal edema by measuring the total retinal thickness using optical coherence tomography (OCT) and the thickness of the inner nuclear layer (INL) by hematoxylin-eosin (H&E) staining were investigated. Oral administration of arctigenin ameliorated retinal edema in an RVO murine model by inhibiting the decrease in occludin and vascular endothelial (VE)-cadherin. Moreover, in retinas with edema, arctigenin suppressed the induction of VEGF, tumor necrosis factor α (TNFα), and matrix metallopeptidase 9 (MMP9). Next, the effects of arctigenin on barrier function were assessed in human retinal microvascular endothelial cells (HRMECs) by measuring the trans-endothelial electrical resistance (TEER) and conducting fluorescein isothiocyanate (FITC)-dextran permeability assays. Arctigenin showed a protective effect against VEGF-induced barrier dysfunction. In addition, arctigenin inhibited the TNFα-mediated activation of the nuclear factor-kappaB (NF-κB)/p38 mitogen-activated protein kinase (MAPK) pathway. These results suggested that oral administration of arctigenin has beneficial effects on retinal edema by inhibiting vascular hyperpermeability in endothelial cells.

Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro

An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent.Key messages• Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis.• The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia.• In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2.• PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.

The Antimicrobial Peptide Cathelicidin Exerts Immunomodulatory Effects via Scavenger Receptors

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). We performed DNA microarray, reverse transcription polymerase chain reaction, immunoblotting, and proximity ligation assays using cultured keratinocytes treated with LL-37 and/or the DAMP poly(I:C), a synthetic double-stranded RNA. In contrast to the combination of LL-37 and poly(I:C), LL-37 alone induced genes related to biological metabolic processes such as VEGFA and PTGS2 (COX-2). Inhibition of FPR2, a known receptor for cathelicidin, partially suppressed the induction of VEGFA and PTGS2. Importantly, VEGFA and PTGS2 induced by LL-37 alone were diminished by the knockdown of scavenger receptors including SCARB1 (SR-B1), OLR1 (SR-E1), and AGER (SR-J1). Moreover, LL-37 alone, as well as the combination of LL-37 and poly(I:C), showed proximity to the scavenger receptors, indicating that LL-37 acts via scavenger receptors and intermediates between them and poly(I:C). These results showed that the broad function of cathelicidin is generally dependent on scavenger receptors. Therefore, inhibitors of scavenger receptors or non-functional mock cathelicidin peptides may serve as new anti-inflammatory and immunosuppressive agents.

Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

During mycobacterial infections, pathogenic mycobacteria manipulate both host immune and stromal cells to establish and maintain a productive infection. In humans, non-human primates, and zebrafish models of infection, pathogenic mycobacteria produce and modify the specialized lipid trehalose 6,6'-dimycolate (TDM) in the bacterial cell envelope to drive host angiogenesis toward the site of forming granulomas, leading to enhanced bacterial growth. Here, we use the zebrafish-Mycobacterium marinum infection model to define the signaling basis of the host angiogenic response. Through intravital imaging and cell-restricted peptide-mediated inhibition, we identify macrophage-specific activation of NFAT signaling as essential to TDM-mediated angiogenesis invivo. Exposure of cultured human cells to Mycobacterium tuberculosis results in robust induction of VEGFA, which is dependent on a signaling pathway downstream of host TDM detection and culminates in NFATC2 activation. As granuloma-associated angiogenesis is known to serve bacterial-beneficial roles, these findings identify potential host targets to improve tuberculosis disease outcomes.

A Novel PHD2/VHL-mediated Regulation of YAP1 Contributes to VEGF Expression and Angiogenesis.

The transcriptional co-activator YAP1 is the major oncogenic component of the Hippo signaling pathway and contributes to the genesis and progression of various tumors, including non-small cell lung cancer (NSCLC). YAP1 levels are regulated by the canonical Hippo kinases, MST1/2 and LATS1/2, which modulate its cytoplasmic retention and proteasomal degradation. While non-canonical regulation of YAP1 has been reported, its role in hypoxic response is not fully elucidated. The studies presented here show that YAP1 levels and function are modulated by VHL and PHD2. YAP1 could regulate multiple genes involved in angiogenesis through E2F1; it also associates with HIF1α in cancer cells under hypoxic conditions, inducing the VEGF-A promoter. Under normoxic conditions, PHD2 associates with and hydroxylates specific proline residues on YAP1, facilitating its interaction with VHL and promoting ubiquitination and subsequent proteasomal degradation. Exposure to hypoxia dissociates YAP1 from PHD2 and VHL, elevating YAP1 levels and enhancing its association with HIF1α. YAP1-HIF1α interaction was higher in NSCLC and RCC samples, indicating a role for this interaction in the genesis of these cancers. Our results thus reveal a novel mode of regulation of YAP1 by PHD2 and VHL in normoxic cells, suggesting that YAP1-mediated induction of VEGF and other genes contributes to hypoxic response in tumors.

A case report of an uncommon presentation of Nephroblastoma (Wilms tumor): in an older child with polycythemia and elevated erythropoietin

Abstract Introduction/Objective Wilms tumor (nephroblastoma), is a neoplasm that has a prevalence of 1 per 10,000 before the age of fifteen, with 50% occurring before age 3. WT1 (11p13) gene, a zinc finger transcription factor, is expressed in early urogenital system development and mutations here represent one of the primary pathways to the development of Wilms tumor. Although mouse model studies have shown that WT1 may be a transcriptional activator of erythropoietin (EPO) gene, increases in serum erythropoietin are rarely seen in patients with Wilms tumor. Concurrent polycythemia is a further rarity with only 11 such reports in literature. This is intriguing since WT1 mutations represent the most common mutation pathway in Wilms Tumor. Interestingly, other tumor types associated with elevated serum EPO and polycythemia (e.g. renal cell carcinoma and metanephric adenoma) are thought to do so through the generation of hypoxia inducible factor, and induction of VEGF. Methods/Case Report Here we present a case of Wilms tumor in a 7-year-old female who was establishing care after moving to Vermont. Physical examination showed possible splenomegaly ultimately discovered to be a large LUQ abdominal mass with a concurrent discovery of polycythemia. Subsequent serum EPO was 308 (Ref: 2.6-18.5 mIU/mL).). The mass was surgically removed with resolution of polycythemia. Histological evaluation showed a triphasic, blastema predominant Wilms tumor with favorable histology. Heterologous, vascular differentiation was seen in the stroma, positive for CD34 and CD31, and negative for D2-40. Results (if a Case Study enter NA) NA Conclusion Based on these findings, this phenomenon may be related to increased VEGF expression resulting in this patient’s increased serum EPO and polycythemia, and heterologous vascular differentiation within the tumor stroma. This is the first report of histology in a case of Wilms tumor associated with high serum EPO and polycythemia and may indicate an alternative pathway for the generation of EPO in Wilms tumor.

"Fasting: An Effective Preconditioning Method to Increase Fat Graft Survival".

Most preconditioning techniques before fat grafting require external manipulation. Since nutrition is the main factor maintaining the balance of lipogenesis and lipolysis, we hypothesized that fasting before undergoing autologous fat grafting may increase lipolysis and reduce adipocyte size, thereby improving the fat graft survival rate. C57BL/6 mice were divided into 24 h starved or fed groups. Adipose tissue lipolysis, adipogenesis, and angiogenesis-related gene expression, in fat from both groups, were analyzed. The volume and weight of the grafted fat at 4-8 weeks postoperatively were measured using micro-computed tomography. Immunohistochemistry staining and mRNA expression analysis were also performed to evaluate the effect of fasting on fat graft survival. Fasting decreased adipocyte size by inducing adipose tissue lipolysis. Adipogenesis-related genes were remarkably downregulated while lipolysis-related genes and angiogenesis inducer genes were significantly upregulated in the starved adipose tissue. The mice grafted with the fat from the 24 h starved group had approximately 20% larger volumes and considerably heavier weights than those from the fed group. Increased viable adipocytes and vessels, and reduced macrophages in the fat grafts obtained from the 24 h starved group were also observed. Fasting for 24 h before harvesting fat increased the retention volume of fat graft by increasing angiogenesis via VEGF induction. Therefore, fasting would be a novel and reliable preconditioning strategy to improve graft survival in autologous fat grafting. 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.

HSP90 acts as a senomorphic target in senescent retinal pigmental epithelial cells.

The senescence of retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD), a leading cause of blindness in the world. HSP90 is a predominant chaperone that regulates cellular homeostasis under divergent physio-pathological conditions including senescence. However, the role of HSP90 in senescent RPE cells still remains unclear. Here, we reported that HSP90 acts as a senomorphic target of senescent RPE cells in vitro. Using H2O2-induced senescent ARPE-19 cells and replicative senescent primary RPE cells from rhesus monkey, we found that HSP90 upregulates the expression of IKKα, and HIF1α in senescent ARPE-19 cells and subsequently controls the induction of distinct senescence-associated inflammatory factors. Senescent ARPE-19 cells are more resistant to the cytotoxic HSP90 inhibitor IPI504 (IC50 = 36.78 μM) when compared to normal ARPE-19 cells (IC50 = 6.16 μM). Administration of IPI504 at 0.5–5 μM can significantly inhibit the induction of IL-1β, IL-6, IL-8, MCP-1 and VEGFA in senescent ARPE-19 and the senescence-mediated migration of retinal capillary endothelial cells in vitro. In addition, we found that inhibition of HSP90 by IPI504 reduces SA-β-Gal’s protein expression and enzyme activity in a dose-dependent manner. HSP90 interacts with and regulates SA-β-Gal protein stabilization in senescent ARPE-19 cells. Taken together, these results suggest that HSP90 regulates the SASP and SA-β-Gal activity in senescent RPE cells through associating with distinctive mechanism including NF-κB, HIF1α and lysosomal SA-β-Gal. HSP90 inhibitors (e.g. IPI504) could be a promising senomorphic drug candidate for AMD intervention.

Abstract 1078: Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features

Abstract Trastuzumab and trastuzumab emtansine (T-DM1) targeted therapies are the main choice for the treatment of HER2-overexpressing breast cancer patients. However, de novo or acquired resistance is still the major obstacle in clinical practice. It has been shown that several pathways, including HER2, can lead to HIF-α stabilization in breast cancer cells. Thus, the purpose of our study was to analyse the effect of hypoxia in acquired resistance to anti-HER2 therapies. We used HER2-overexpressing BT-474 and non-overexpressing MCF-7 human breast cancer cell lines. As an acute hypoxia model, we added CoCl2 (100 µM) to cell culture medium. The hypoxic status of the cells was evaluated by a Western blot analysis, showing a peak of HIF-1α expression after 6 hours of CoCl2 exposure. This result correlated with VEGF induction, as measured by RT-qPCR (p<0.05). Interestingly, under hypoxia, BT-474 cells treated with increasing concentrations of trastuzumab and T-DM1 for 72 hours presented a significantly higher viability compared with non-hypoxic cells (p<0.05). However, although the drugs decreased membrane HER2 protein expression (p<0.01), HER2 levels did not change under hypoxic conditions, as shown by flow cytometry and Western blot assays. Moreover, drug effects on MCF-7 cells did not change under hypoxic conditions. To further analyse this cytoprotective effect of hypoxia, proliferation-associated markers such as PCNA expression and AKT activation were evaluated by Western blot, while apoptosis frequency was measured by flow cytometry in BT-474 cells. PCNA and pAKT expression remained unaltered, while T-DM1-mediated apoptosis was significantly reduced under hypoxia (p<0.05). Then, using different open gene expression datasets, we identified a group of four genes (COL12A1, DKK1, CEACAM5 and ANTXR1) differentially expressed in T-DM1 resistant HER2-overexpressing breast cancer BT-474 and KPL-4 cell lines (p<0.05), which were coexpressed with hypoxia-related genes (HIF1A, EPAS1 and VEGFA) in HER2-overexpressing breast cancer tumours and cell lines from TCGA (p<0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p<0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p<0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p<0.05), showing a tendency towards a cancer stem cell phenotype. It is known that hypoxia represents a common feature of metastatic tumours and has a role in regulating stem cell behaviour. According to this, our results highlight a link between anti-HER2 therapy resistance and epithelial-to-mesenchymal transition characteristics, where hypoxia plays a key role which deserves further study. Citation Format: Virginia Judith Wolos, Ivana Jaqueline Tapia, Marianela Abrigo, Ezequiel Lacunza, Elisa Dora Bal de Kier Joffé, Gabriel León Fiszman. Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1078.

Propranolol Participates in the Treatment of Infantile Hemangioma by Inhibiting HUVECs Proliferation, Migration, Invasion, and Tube Formation.

Objective Infantile hemangiomas (IHs) are the most common benign tumors in infancy. The purpose of this study was to study the effects of propranolol on the function of human umbilical vein endothelial cells (HUVECs), in order to preliminarily elucidate the mechanism of propranolol in the treatment of IHs. Methods HUVECs were treated with different concentrations of propranolol (30 μM, 60 μM, 90 μM, and 120 μM) with or without VEGF. Their proliferation, migration, invasion, adhesion, and tube formation ability were tested by using CCK-8, wound healing assay, transwell, cell adhesion assay, and tube formation assay. The expressions of HUVECs angiogenesis signaling molecules pERK/ERK, pAKT/AKT, p-mTOR/mTOR, and pFAK/FAK were detected by Western blot. Results Compared with the control group, propranolol could significantly inhibit the proliferation, migration, invasion, adhesion, and tube formation of HUVECs. Further studies showed that it could not only inhibit the migration, invasion, and tube formation ability of HUVECs after VEGF induction but also inhibit the phosphorylated protein expressions of angiogenesis-related signaling molecules like AKT, mTOR, ERK, and FAK in HUVECs, with a concentration-dependent inhibitory effect. Conclusion Propranolol can inhibit the proliferation, migration, invasion, adhesion, and tube formation of hemangioma endothelial cells; block VEGF-mediated angiogenesis signaling pathway; suppress the expressions of downstream angiogenesis-related signaling molecules; and ultimately achieve the effect of treatment of IHs.

Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts.

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

Does survivin overexpression enhance the efficiency of fibroblast cell-based wound therapy?

Cell-based wound therapy is faced with some limiting factors that decrease the therapeutic efficacy of transplanted cells. In this study, we aimed to genetically modify fibroblast cells with anti-apoptotic Survivin gene(Birc5) before cell transplantation. In vitro, pIRES2-eGFP-Survivin plasmid was transfected into the fibroblast cells and the growth curve was evaluated for transfected and normal cells performing MTT assay. In vivo, two 6-diameter cutaneous wounds were created at mice dorsal skin. Fibrin clot was used as a delivery vehicle to transfer cells into the wound bed. The effects of four treatment groups including (a) Cell-SVV-Clot (b) Cell-GFP-Clot, (c) Normal cell-Clot and, (d) Clot alone were evaluated. After 1,2,3,7 and 14days post-transplantation, the wounds were photographed for evaluating the wound closure rate and wound samples were obtained. Angiogenesis and formation of granulated tissue were assessed via H&E staining for wound samples. The expression levels of Survivin, VEGF, and bFGF genes were also determined using qRT-PCR. The MTT assay showed similar proliferation potential of transfected cells with normal cells verifying that Survivin had no detrimental effect. Compared to the Normal cell-Clot group, the Survivin overexpression was seen for 3days in the Cell-SVV-Clot group verifying the cell survival during the early stage of wound healing. The Survivin further upregulated VEGF and bFGF expressions resulting in more angiogenesis and formation of granulated tissue by day 3 and 14. The treated wounds with Cell-SVV-Clot were regenerated with a higher wound closure rate by day 7 compared to Normal cell-Clot and Clot groups. Survivin enhanced wound healing through induction of VEGF and bFGF at particular times post-wounding that led to a more structured-epidermis with higher angiogenesis and granulation tissue formation rate.

THE 30 YEARS' CLINICAL EXPERIENCE IN DIAGNOSIS AND TREATMENT OF ENDOMETRIAL OVARIAN CYSTS IN PATIENTS OF REPRODUCTIVE AGE

Endometriosis occurs in 50 % of women with infertility. Ovarian endometriosis affects 55 % of women with this disease. Surgical treatment of endometrium is indicated if its size is over 3 cm. After removal of the endometriomas there are used diathermic, bipolar energy, radio wave energy, argon plasma coagulation for hemostasis of ovarian tissue. The use of argon plasma coagulation in ovarian hemostasis causes not only hemostatic but also protective effect, which is mediated by the induction of HSP27, SOD2, VEGF and iNOS, which renders an organ−preserving effect when applied to ovarian tissues. Repeated surgery for ovarian endometrium should be performed taking into account the risk of reduced levels of antimullerian hormone, which may adversely affect the further implementation of reproductive function, including the use of assisted reproductive technologies. The authors comparatively have evaluated the treatment of 900 patients of reproductive age with endometrioid cysts of the ovaries and other concomitant gynecological pathology, which were three clinical groups. The patients received radiowave energy, argonoplasmic coagulation, intraoperative use of various anti−adhesive drugs and combinations of drugs in the postoperative period. It was found that the use of argon plasma coagulation for hemostasis of ovarian tissues after removal of endometrioid cysts, polyethylene oxide gel with carboxymethylcellulose intraoperatively and gonadotropin−releasing hormone agonists, dienogest 2 mg per day for three months contributed to the pregnancy onset in post−srugery period in 56.7 % of patients. It has been noted that in women the presence of adhesions in combination with corpus luteum cysts, endometrial polyps, uterine leiomyoma reduces the onset of pregnancy by 8.0, 12, and 24 times, respectively. Key words: endometrial ovarian cysts, diagnosis, treatment, rehabilitation in the post−surgery period.

Human Umbilical Cord Mesenchymal Stem Cell Differentiation Into Odontoblast-Like Cells and Endothelial Cells: A Potential Cell Source for Dental Pulp Tissue Engineering.

ObjectivesDental pulp regeneration is considered an ideal approach for treating dental pulp disease. Because pulp is composed of various cells, determining the proper seed cells is critical. We explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration.MethodsLiquid extract of human treated dentin matrix (LE-TDM) was acquired to culture hUCMSCs. Odontoblast-specific markers were detected by western blot, qRT-PCR, and immunofluorescence assays. Endothelial differentiation of hUCMSCs was examined according to VEGF induction by western blot, qRT-PCR, and Matrigel assays. hUCMSCs and VEGF-induced hUCMSCs (V-hUCMSCs) were also cocultured in vivo for the Matrigel plug assay and in vitro for RNA-sequencing (RNA-seq). Finally, encapsulated mono-cultured hUCMSCs or cocultured hUCMSCs and V-hUCMSCs in scaffolds were injected into the root segments and transplanted into immunodeficient mice for dental pulp regeneration.ResultsUnder LE-TDM induction, hUCMSCs expressed specific odontoblast markers (DSPP, DMP-1, DSP). Under VEGF induction, hUCMSCs expressed functional endothelial markers (CD31, eNOs, vWF). In vivo, the Matrigel plug assay indicated that cocultured hUCMSCs and V-hUCMSCs formed extensive vessel-like structures. RNA-seq results indicated that cocultured V-hUCMSCs exhibited high Hif-1 signaling pathway activity. Both the hUCMSCs mono-culture and coculture groups showed pulp-like tissue regeneration. The cocultured group showed more extracellular matrix and vascularization than the mono-cultured group in vivo.ConclusionhUCMSCs can differentiate into odontoblast-like cells and functional endothelial cells. Cocultured hUCMSCs and V-hUCMSCs formed vessel-like structures and regenerated dental pulp-like tissue. Therefore, hUCMSCs can be used as an alternative seed cell source for angiogenesis and dental pulp regeneration.

Eukaryotic elongation factor-2 kinase (eEF2K) signaling in tumor and microenvironment as a novel molecular target

Eukaryotic elongation factor-2 kinase (eEF2K), an atypical member of alpha-kinase family, is highly overexpressed in breast, pancreatic, brain, and lung cancers, and associated with poor survival in patients. eEF2K promotes cell proliferation, survival, and aggressive tumor characteristics, leading to tumor growth and progression. While initial studies indicated that eEF2K acts as a negative regulator of protein synthesis by suppressing peptide elongation phase, later studies demonstrated that it has multiple functions and promotes cell cycle, angiogenesis, migration, and invasion as well as induction of epithelial-mesenchymal transition through induction of integrin β1, SRC/FAK, PI3K/AKT, cyclin D1, VEGF, ZEB1, Snail, and MMP-2. Under stress conditions such as hypoxia and metabolic distress, eEF2K is activated by several signaling pathways and slows down protein synthesis and helping cells to save energy and survive. In vivo therapeutic targeting of eEF2K by genetic methods inhibits tumor growth in various tumor models, validating it as a potential molecular target. Recent studies suggest that eEF2K plays a role in tumor microenvironment cells by monocyte chemoattractant protein-1 (MCP-1) and accumulation of tumor-associated macrophages. Due to its clinical significance and the pivotal role in tumorigenesis and progression, eEF2K is considered as an important therapeutic target in solid tumors. However, currently, there is no specific and potent inhibitor for translation into clinical studies. Here, we aim to systematically review current knowledge regarding eEF2K in tumor biology, microenvironment, and development of eEF2K targeted inhibitors and therapeutics.

Co-delivery of paclitaxel and anti-VEGF siRNA by tripeptide lipid nanoparticle to enhance the anti-tumor activity for lung cancer therapy

The combination of chemotherapeutic drug paclitaxel (PTX) and VEGF siRNA could inhibit cancer development with synergistic efficacy. However, efficient and safe delivery systems with high encapsulation efficiency of PTX and a long-time release of drugs are urgently needed. In this study, novel nanoparticles (PTX/siRNA/FALS) were constructed by using tripeptide lipid (L), sucrose laurate (S), and folate-PEG2000-DSPE (FA) to co-deliver PTX and siRNA. The cancer cell targeting nanoparticle carrier (PTX/siRNA/FALS) showed anticipated PTX encapsulation efficiency, siRNA retardation ability, improved cell uptake and sustained and controlled drug release. It led to significant anti-tumor activity in vitro and in vivo by efficient inhibition of VEGF expression and induction of cancer cell apoptosis. Importantly, the biocompatibility of the carriers and low dosage of PTX required for effective therapy greatly reduced the toxicity to mice. The targeting nanoparticles show potential as an effective co-delivery platform for RNAi and chemotherapy drugs, aiming to improve the efficacy of cancer therapy.

LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

One of the health hazards of PM2.5 exposure is to induce pulmonary inflammatory responses. In our previous study, we demonstrated that exposing both the immortalized and primary human bronchial epithelial cells to PM2.5 results in a significant upregulation of VEGF production, a typical signaling event to trigger chronic airway inflammation. Further investigations showed that PM2.5 exposure strongly induces ATR/CHK1/p53 cascade activation, leading to the induction of DRAM1-dependent autophagy to mediate VEGF expression by activating Src/STAT3 pathway. In the current study, we further revealed that TIGAR was another transcriptional target of p53 to trigger autophagy and VEGF upregulation in Beas-2B cells after PM2.5 exposure. Furthermore, LKB1, but not ATR and CHK1, played a critical role in mediating p53/TIGAR/autophagy/VEGF pathway activation also by linking to Src/STAT3 signaling cascade. Therefore, on combination of the previous report, we have identified both ATR/CHK1/p53/DRAM1- and LKB1/p53/TIGAR- dependent autophagy in mediating VEGF production in the bronchial epithelial cells under PM2.5 exposure. Moreover, the in vivo study further confirmed VEGF induction in the airway potentially contributed to the inflammatory responses in the pulmonary vascular endothelium of PM2.5-treated rats. Therefore, blocking VEGF expression or autophagy induction might be the valuable strategies to alleviating PM2.5-induced respiratory injuries.

Hypoxia and EGF Stimulation Regulate VEGF Expression in Human Glioblastoma Multiforme (GBM) Cells by Differential Regulation of the PI3K/Rho-GTPase and MAPK Pathways.

Glioblastoma multiforme (GBM) is one of the most common and deadly cancers of the central nervous system (CNS). It is characterized by the presence of hypoxic regions, especially in the core, leading to an increase in vascularity. This increased vascularization is driven by the expression of the major angiogenic inducer VEGF and the indirect angiogenic inducer Epidermal growth factor (EGF), which stimulates VEGF expression. In this study, we examine the regulation of VEGF by both hypoxia and the EGF signaling pathway. We also examine the involvement of pathways downstream from EGF signaling, including the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway and the Phosphatidylinositol-3-kinase/RhoA/C (PI3K/RhoA/C) pathway in this regulation. Our results show that VEGF expression and secretion levels increase following either hypoxia or EGF stimulation, with the two stimuli signaling in parallel. We also observed an increase in ERK and protein kinase B (Akt) phosphorylation, in response to EGF stimulation, with kinetics that correlated with the kinetics of the effect on VEGF. Using pharmacological inhibitors against ERK and PI3K and small interfering RNAs (siRNAs) against RhoA and RhoC, we found that both the ERK and the PI3K/RhoA/C pathways have to cooperate in order to lead to an increase in VEGF expression, downstream from EGF. In response to hypoxia, however, only ERK was involved in the regulation of VEGF. Hypoxia also led to a surprising decrease in the activation of PI3K and RhoA/C. Finally, the decrease in the activation of these Rho-GTPases was found to be mediated through a hypoxia-driven overexpression of the Rho-GTPase GTPase activating protein (GAP), StarD13. Therefore, while under normoxic conditions, EGF stimulates the activation of both the PI3K and the MAPK pathways and the induction of VEGF, in glioblastoma cells, hypoxic conditions lead to the suppression of the PI3K/RhoA/C pathway and an exclusive switch to the MAPK pathway.

Endothelium-dependent molecular mechanisms of articular cartilage and subchondral bone remodeling in conditions of cardiovascular comorbidity

Aim. To determine the expression level of endothelial factors (VEGF-A, CD34 + ) in the tissues of the joints in experimental arterial hypertension (AH), hyperlipidemia (HL) and their combination. Material and methods . An experimental study was conducted on 24 sexually mature males of outbred guinea pigs. All animals were divided into 4 groups of 6 animals each. In the first group, AH was reproduced by intramuscular injection of a hydrocartisone solution. HL in the second group was simulated by intraperitoneal injection 1 time in 3 weeks of Tween 80 and diet modification. In the third group, the combination of AH and GL was reproduced by injection of a hydrocartisone solution, Tween-80 and diet modification. On the 60-day animals were pull out of the experiment by euthanasia. Hock joint tissue sampling was performed, in which the expression of endothelial vascular growth factor A (VEGF-A), CD34 was determined using a peroxidase immunohistochemical reaction. Results. It was found that during experimental AH, expression of VEGF-A increased in the subchondral bone. In experimental HL, preferential induction of VEGF-A and CD34 in the articular cartilage and subchondral bone is observed. When combined with cardiovascular factors, the highest expression of the molecular mechanisms of cardiometabolic stress, including VEGF-A and CD34 is observed. Conclusion. The results indicate that cardiovascular factors affect the joint tissue. This allows us to make an assumption that the joint tissues belong to the target organs of cardiovascular diseases and make it possible to judge the important role of cardiovascular comorbidity in the pathogenesis of degenerative inflammatory joint diseases.

Vitamin D: A New Perspective in Treatment of Cerebral Vasospasm After Subarachnoid Hemorrhage

Abstract INTRODUCTION Cerebral vasospasm (CVS) is a severe complication after subarachnoid hemorrhage (SAH). We explored vitamin D (VitD) as a possible therapeutic option for CVS. METHODS Between 2007 and 2015, 25-vitaminD3 (VitD3) levels and data of SAH patients admitted during the months with a peak vs nadir of VitD3-values were analyzed, retrospectively. Furthermore, VitD3 and vasospasm/outcome data in SAH patients admitted in 2017 were correlated prospectively. A mice SAH model and cell culture studies were used to determine the involved mechanisms of 1,25-dihydroxy-vitaminD3. RESULTS A significant lower vasospasm rate and improved outcome in SAH patients admitted in summer as compared to winter time was identified by the retrospective analysis. In the SAH mouse model, active 1,25-VitD3 attenuated CVS development, as determined by vessel diameter of basilar artery (BA) and reduced neurological deficit. 1,25-VitD3 attenuated the inflammation of the BA in response to blood. Deletion of the myeloid or endothelial VitD-receptor decreased the protective 1,25-VitD3 effect. Co-culture of myeloid and endothelial cells with blood confirmed the anti-inflammatory effect of 1,25-VitD3 and revealed a selective induction of SDF1a, VEGF, and eNOS. In mice, SDF1a mimicked the protective 1,25-VitD3 effect. In patients, high VitD3 levels were associated with a significant higher rate of favorable outcome, prospectively. CVS severity was inversely correlated with the VitD level. Patients with severe CVS exhibited attenuated expression of SDF1a on myeloid cells. CONCLUSION 1,25-VitD3 attenuates CVS after SAH. VitD chould be tested as potential treatment option to prevent CVS after SAH.