Formation Of Tubular Structure Research Articles

Antitumor activity of anlotinib in malignant melanoma: modulation of angiogenesis and vasculogenic mimicry.

Malignant melanoma presents a formidable challenge due to its aggressive metastatic behavior and limited response to current treatments. To address this, our study delves into the impact of anlotinib on angiogenesis and vasculogenic mimicry using malignant melanoma cells and human umbilical vein endothelial cells. Evaluating tubular structure formation, cell proliferation, migration, invasion, and key signaling molecules in angiogenesis, we demonstrated that anlotinib exerts a dose-dependent inhibition on tubular structures and effectively suppresses cell growth and invasion in both cell types. Furthermore, in a mouse xenograft model, anlotinib treatment resulted in reduced tumor growth and vascular density. Notably, the downregulation of VEGFR-2, FGFR-1, PDGFR-β, and PI3K underscored the multitargeted antitumor activity of anlotinib. Our findings emphasize the therapeutic potential of anlotinib in targeting angiogenesis and vasculogenic mimicry, contributing to the development of novel strategies for combating malignant melanoma.

MiR-455–3p regulates lymphangiogenesis in silicosis by regulating VEGF-C/VEGFR3

Silicosis is a disease characterized by lung inflammation and fibrosis caused by long-term inhalation of free silicon dioxide (SiO2). Recent studies have found that a large number of lymphatic hyperplasia occurs during the occurrence and development of silicosis. miRNAs play an important role in lymphangiogenesis. However, the regulation and mechanism of miRNAs on lymphangiogenesis in silicosis remain unclear. In this study, lymphangiogenesis was observed in silicosis rats, and VEGF-C-targeted miRNAs were screened, and the effect of miRNAs on the formation of human lymphatic endothelial cells (HLECs) tubular structure was investigated in vitro. The results showed that SiO2 promoted the expressions of Collagen Ι and α-SMA, TNF-α, IL-6 and VEGF-C increased first and then decreased, and promoted the formation of lymphatic vessels. Bioinformatics methods screened miR-455–3p for targeted binding to VEGF-C, and dual luciferase reporter genes confirmed VEGF-C as the target gene of miR-455–3p, and miR-455–3p was down-regulated in the lung tissue of silicosis rats. Transfection of miR-455–3p Inhibitors down-regulated the expression level of miR-455–3p and up-regulated the expression levels of VEGF-C and VEGFR-3 in HLECs, enhanced migration ability and increased tube formation. Transfection of miR-455–3p Mimics showed an opposite trend. These results suggest that miR-455–3p further regulates the tubular structure formation of HLECs by regulating VEGF-C/VEGFR3. Therefore, targeting miR-455–3p may provide a new therapeutic strategy for SiO2-induced silicosis injury.

Multifunctional Exosomes Derived from M2 Macrophages with Enhanced Odontogenesis, Neurogenesis and Angiogenesis for Regenerative Endodontic Therapy: An In Vitro and In Vivo Investigation.

Exosomes derived from M2 macrophages (M2-Exos) exhibit tremendous potential for inducing tissue repair and regeneration. Herein, this study was designed to elucidate the biological roles of M2-Exos in regenerative endodontic therapy (RET) compared with exosomes from M1 macrophages (M1-Exos). The internalization of M1-Exos and M2-Exos by dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) was detected by uptake assay. The effects of M1-Exos and M2-Exos on DPSC and HUVEC behaviors, including migration, proliferation, odonto/osteogenesis, neurogenesis, and angiogenesis were determined in vitro. Then, Matrigel plugs incorporating M2-Exos were transplanted subcutaneously into nude mice. Immunostaining for vascular endothelial growth factor (VEGF) and CD31 was performed to validate capillary-like networks. M1-Exos and M2-Exos were effectively absorbed by DPSCs and HUVECs. Compared with M1-Exos, M2-Exos considerably facilitated the proliferation and migration of DPSCs and HUVECs. Furthermore, M2-Exos robustly promoted ALP activity, mineral nodule deposition, and the odonto/osteogenic marker expression of DPSCs, indicating the powerful odonto/osteogenic potential of M2-Exos. In sharp contrast with M1-Exos, which inhibited the neurogenic capacity of DPSCs, M2-Exos contributed to a significantly augmented expression of neurogenic genes and the stronger immunostaining of Nestin. Consistent with remarkably enhanced angiogenic markers and tubular structure formation in DPSCs and HUVECs in vitro, the employment of M2-Exos gave rise to more abundant vascular networks, dramatically higher VEGF expression, and widely spread CD31+ tubular lumens in vivo, supporting the enormous pro-angiogenic capability of M2-Exos. The multifaceted roles of M2-Exos in ameliorating DPSC and HUVEC functions potentially contribute to complete functional pulp-dentin complex regeneration.

Uncovering the Anti-Angiogenic Mechanisms of Centella asiatica via Network Pharmacology and Experimental Validation.

Centella asiatica (CA) has been used to address cancer for centuries in traditional Chinese medicine (TCM). Previous studies demonstrated its anti-angiogenesis efficacy, but the underlying mechanism of its action remains to be further clarified. This study aims to investigate the underlying mechanisms of CA and its triterpenes in anti-angiogenesis for cancer therapeutics through network pharmacology and experimental validation. Cytoscape was used to construct a network of compound-disease targets and protein-protein interactions (PPIs) from which core targets were identified. GO and KEGG analyses were performed using Metascape, and the AutoDock-Vina program was used to realize molecular docking for further verification. Then, VEGF165 was employed to establish an induced angiogenesis model. The anti-angiogenic effects of CA were evaluated through assays measuring cell proliferation, migration, and tubular structure formation. Twenty-five active ingredients in CA had potential targets for anti-angiogenesis including madecassoside, asiaticoside, madecassic acid, asiatic acid, and asiaticoside B. In total, 138 potential targets for CA were identified, with 19 core targets, including STAT3, SRC, MAPK1, and AKT1. A KEGG analysis showed that CA is implicated in cancer-related pathways, specifically PD-1 and AGE-RAGE. Molecular docking verified that the active components of CA have good binding energy with the first four important targets of angiogenesis. In experimental validation, the extracts and triterpenes of CA improved VEGF165-induced angiogenesis by reducing the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Our results initially demonstrate the effective components and great anti-angiogenic activity of CA. Evidence of the satisfactory anti-angiogenic action of the extracts and triterpenes from CA was verified, suggesting CA's significant potential as a prospective agent for the therapy of cancer.

Self-templating fabrication of Ti-doped SnO2 @carbon nanotubes via electrospinning for high-performance lithium-ion batteries

Tin dioxide (SnO2) has attracted extensive attention as an anode for lithium-ion batteries (LIBs) due to its high theoretical specific capacity, however, it suffers from the severe volume changes upon lithiation/delithiation cycling, thus leading to fast capacity fading. To overcome the drawbacks, we herein report a facile one-pot self-templating fabrication of Ti-doped SnO2 @carbon nanotubes (Ti-SnO2 @CNTs), via annealing the electrospun precursor nanofibers of tetrabutyl titanate (TBT), tetraethyl orthosilicate (TEOS), polyvinylidene difluoride (PVDF), polyvinylpyrrolidone (PVP), and stannous chloride (SnCl2). Interestingly, the introduction of TBT or not can lead to the controlled fabrication of the tubular or solid fibrous structure. During the annealing process, the chemical reaction between PVDF, TEOS, and TBT in-situ leads to the formation of tubular structure, while the SnCl2 converts into ultrafine SnO2 nanoparticles, embedding into the PVP-derived N-doped carbon nanotubes. When employed as anode materials for LIBs, the Ti-SnO2 @CNTs deliver high reversible capacities of 868.6/495.9 mAh g−1 at 0.2/1.0 A g−1 after 200/500 cycles, higher than those for the fibrous SnO2 @CNFs counterpart (654.9/391.08 mAh g−1). The better lithium storage performance is related to the unique carbon nanotube structure and the Ti-doping, which bring improved electrical conductivity, enhanced structural stability, and shortened ion diffusion path. More importantly, we demonstrate the facile electrospinning fabrication of tubular nanostructured hybrid electrodes for high-performance energy storage applications.

Site-specific periosteal cells with distinct osteogenic and angiogenic characteristics.

This study aimed to investigate the site-specific characteristics of rat mandible periosteal cells (MPCs) and tibia periosteal cells (TPCs) to assess the potential application of periosteal cells (PCs) in bone tissue engineering (BTE). MPCs and TPCs were isolated and characterized. The potential of proliferation, migration, osteogenesis and adipogenesis of MPCs and TPCs were evaluated by CCK-8, scratch assay, Transwell assay, alkaline phosphatase staining and activity, Alizarin Red S staining, RT‒qPCR, and Western blot (WB) assays, respectively. Then, these cells were cocultured with human umbilical vein endothelial cells (HUVECs) to investigate their angiogenic capacity, which was assessed by scratch assay, Transwell assay, Matrigel tube formation assay, RT‒qPCR, and WB assays. MPCs exhibited higher osteogenic potential, higher alkaline phosphatase activity, and more mineralized nodule formation, while TPCs showed a greater capability for proliferation, migration, and adipogenesis. MPCs showed higher expression of angiogenic factors, and the conditioned medium of MPCs accelerated the migration of HUVECs, while MPC- conditioned medium induced the formation of more tubular structure in HUVECs in vitro. These data suggest that compared to TPCs, MPCs exert more consequential proangiogenic effects on HUVECs. PCs possess skeletal site-specific differences in biological characteristics. MPCs exhibit more eminent osteogenic and angiogenic potentials, which highlights the potential application of MPCs for BTE. Autologous bone grafting as the main modality for maxillofacial bone defect repair has many limitations. Constituting an important cell type in bone repair and regeneration, MPCs show greater potential for application in BTE, which provides a promising treatment option for maxillofacial bone defect repair.

Opening of Cx43-formed hemichannels mediates the Ca2+ signaling associated with endothelial cell migration

Endothelial cell migration is a key process in angiogenesis. Progress of endothelial cell migration is orchestrated by coordinated generation of Ca2+ signals through a mechanism organized in caveolar microdomains. Connexins (Cx) play a central role in coordination of endothelial cell function, directly by cell-to-cell communication via gap junction and, indirectly, by the release of autocrine/paracrine signals through Cx-formed hemichannels. However, Cx hemichannels are also permeable to Ca2+ and Cx43 can be associated with caveolin-1, a structural protein of caveolae. We proposed that endothelial cell migration relies on Cx43 hemichannel opening. Here we show a novel mechanism of Ca2+ signaling in endothelial cell migration. The Ca2+ signaling that mediates endothelial cell migration and the subsequent tubular structure formation depended on Cx43 hemichannel opening and is associated with the translocation of Cx43 with caveolae to the rear part of the cells. These findings indicate that Cx43 hemichannels play a central role in endothelial cell migration and provide new therapeutic targets for the control of deregulated angiogenesis in pathological conditions such as cancer.

Inhibition of AIF-1 alleviates laser-induced macular neovascularization by inhibiting endothelial cell proliferation via restrained p44/42 MAPK signaling pathway

Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.

Angiogenic properties of glial progenitor cells derived from human induced pluripotent stem cells

Diseases associated with impaired blood supply to the brain ranks second term of mortality in the world, losing the place only to coronary heart disease. The incidence of this disease in the world remains high and increasing significantly with the age. The recent years special attention has been paid to the search for new methods of therapy for ischemic diseases, such as study of angiogenic properties of stem cells and their conditioned medium. The aim of this work is studying the angiogenic properties of glial progenitor cells derived from human induced pluripotent stem cells. The study was carried out by testing the proliferative activity, mobility, migration of endothelial cells line EA.hy926 under the influence of glial progenitor cells and their conditioned medium. Also the research was conducted by ability to formation of the tubular and capillary-like structure by cells line EA.hy926 by modeling angiogenesis in the basement membrane matrix in vitro. The conditioned medium obtained by glial progenitor cells at concentrations of total protein 1 and 5 g/ml has a positive influence on the proliferative activity and mobility of the endothelial cells line EA.hy926. At the same time it does not accelerate the formation of the primary tubular and capillary-like structure by the modeling angiogenesis in the basement membrane matrix in vitro. But glial progenitor cells contribute to the formation of tubular and capillary-like structure due to contact-dependent signaling between the two cell types. The primary formed tubular structure had a long processes and large branch points under co cultivation with glial progenitor cells. Sprouting centers also had long and more convoluted processes and large cell clusters during the formation of a capillary-like structure. The glial progenitor cells and their conditioned medium had a positive effect on endothelial cell migration. This effect probably indicated by the production of substances by glial progenitor cells which was chemoattractants for endothelial cells line EA.hy926.

Microstructural evolution mechanism of plain-woven SiC/SiC during thermal ablation

The ablation characteristics of plain-woven SiC/SiC composites were investigated using an oxy-acetylene flame to reveal the failure process. The results showed that the linear ablation rate decreased linearly with time to 0.035 mm/s at 60 s of ablation. The ablation process of single SiC fibers resulted in the formation of a characteristic SiO2 broken porous tubular structure on the external surface. Due to the characteristics of the warp and weft weave structure of the material, resulting in the formation of "micro-pits" on the surface of the SiC fibers.

Antitumor Potential of Withanolide Glycosides from Ashwagandha (Withania somnifera) on Apoptosis of Human Hepatocellular Carcinoma Cells and Tube Formation in Human Umbilical Vein Endothelial Cells.

Hepatocellular carcinoma (HCC) is the fastest-growing tumor capable of spreading to other organs via blood vessels formed by endothelial cells. Apoptosis and angiogenesis-targeting therapies are attractive for cancer treatment. In this study, we aimed to study the in vitro cytotoxicity of Withania somnifera against human HCC (HepG2) cells, identify potential antitumoral withanolide glycosides from the active fraction, and elucidate cytotoxic molecular mechanisms of identified bioactive compounds. W. somnifera (Solanaceae), well-known as ‘ashwagandha’, is an Ayurvedic medicinal plant used to promote health and longevity, and the MeOH extract of W. somnifera root exhibited cytotoxicity against HepG2 cells during initial screening. Bioactivity-guided fractionation of the MeOH extract and subsequent phytochemical investigation of the active n-BuOH-soluble fraction resulted in the isolation of five withanolide glycosides (1–5), including one new metabolite, withanoside XIII (1), aided by liquid chromatography–mass spectrometry-based analysis. The new compound structure was determined by 1D and 2D nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectroscopy, electronic circular dichroism, and enzymatic hydrolysis. In addition, withanoside XIIIa (1a) was identified as the new aglycone (1a) of 1. Isolated withanolide glycosides 1–5 and 1a were cytotoxic toward HepG2 cells; withagenin A diglucoside (WAD) (3) exhibited the most potent cytotoxicity against HepG2 cells, with cell viability less than 50% at 100 μM. WAD cytotoxicity was mediated by both extrinsic and intrinsic apoptosis pathways. Treatment with WAD increased protein expression levels of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Bcl-2-associated X protein (Bax), and cleaved poly(ADP-ribose) polymerase (cleaved PARP) but decreased expression levels of B-cell lymphoma 2 (Bcl-2). Moreover, WAD inhibited tubular structure formation in human umbilical vein endothelial cells (HUVECs) by inhibiting the protein expression of vascular endothelial growth factor receptor 2 and its downstream pathways, including extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). These effects were also enhanced by co-treatment with ERK and PI3K inhibitors. Overall, these results indicate that WAD (3) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers.

Inhibitory effect of ginsenglactone A from Panax ginseng on the tube formation of human umbilical vein endothelial cells and migration of human ovarian cancer cells

BackgroundHere, we aimed to assess the inhibitory effect of a new compound from Panax ginseng on the migration of human ovarian cancer cells and tube formation of human umbilical vein endothelial cells (HUVECs). MethodsA new compound, ginsenglactone A (1), was isolated from ginseng roots, together with seven known compounds (2-8). Spectroscopic data were used to elucidate the chemical structure of 1. The tubular structure formation in HUVECs was assessed by Mayer's hematoxylin staining. The migration of A2780 cells was evaluated using the scratch wound healing assay. ResultsHUVECs treated with 1 had the statistically significant decrease in tubular structure formation compared to the HUVECs treated with compounds 2-8. This effect was enhanced by co-treatment with inhibitors for phosphatidylinositol 3-kinase (PI3K) (LY294002) and extracellular signal-regulated kinase (ERK) (U0126). Treatment with 1 decreased the expression of phosphorylation of ERK, PI3K, vascular endothelial growth factor receptor2 (VEGFR2), Akt, and mammalian target of rapamycin (mTOR). In addition, the ability of A2780 cells to cover the scratched area were also decreased. This effect was enhanced by co-treatment with U0126. Lastly, treatment with 1 decreased the phosphorylation of ERK, matrix metalloproteinase-9 (MMP-9), and MMP-2. ConclusionThese results suggest that ginsenglactone A is a potential inhibitor of HUVEC tubular structure formation and A2780 cellular migration, which may be helpful for understanding its anticancer mechanism.

Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors.

Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.

Preclinical anti‐angiogenic and anti‐cancer activities of BAY1143269 in glioblastoma via targeting oncogenic protein expression

Glioblastoma angiogenesis is critical for tumor growth, making it an appealing target for treatment development. BAY1143269 is a novel inhibitor of mitogen‐activated protein kinase interacting serine/threonine‐protein kinase 1 (MKN1) and has potent anti‐cancer activity. We identified BAY1143269 as an angiogenesis inhibitor, by in vitro and in vivo glioblastoma angiogenesis models. BAY1143269 inhibited the capillary network formation of glioblastoma microvascular endothelial cells (GMECs), particularly the early stage of tubular structure formation. It also inhibited migration and proliferation, and induced apoptosis of GMECs isolated from glioblastoma patients. We found that BAY1143269 acted on GMECs by suppressing the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E‐mediated expression of oncogenic proteins, including those involved in cell cycle, epithelial‐mesenchymal transition (EMT), and pro‐survival. In addition, BAY1143269 suppressed eIF4E phosphorylation, inhibited proliferation, and induced apoptosis of glioblastoma cells. Interestingly, it reduced vascular endothelial growth factor (VEGF) level in tumor cells and culturing medium, demonstrating the inhibitory effect of BAY1143269 on tumor proangiogenic microenvironment. We finally challenged BAY1143269 on the glioblastoma xenograft mice model and observed a significant tumor growth reduction without toxicity in mice receiving oral BAY1143269. Immunoblotting analysis demonstrated significantly less phosphorylated‐eIF4E (p‐eIF4E), cluster of differentiation 31 (CD31) (microvascular endothelial cell marker), and VEGF in tumors from drug‐treated mice. In summary, the inhibition of glioblastoma angiogenesis with BAY1143269 may provide an alternative approach for anti‐glioblastoma therapy.

Quinacrine is active in preclinical models of glioblastoma through suppressing angiogenesis, inducing oxidative stress and activating AMPK

The poor prognosis of glioblastoma requires new innovative treatment strategies. We and others have shown that targeting tumor as well as angiogenesis in glioblastoma are effective therapeutic strategies. In line with these efforts, this work reveals that Quinacrine, an antimalarial drug, is a dual inhibitor of angiogenesis and glioblastoma. Using multiple glioblastoma cell lines, we found that Quinacrine inhibited proliferation and induced apoptosis in these cells, and acted in synergy with Temozolomide. Quinacrine potently inhibited tubular structure formations of glioblastoma microvascular endothelial cell (GMVEC) isolated from glioblastoma patients, especially for early stage tubular structure formation. Although Quinacrine induces apoptosis in GMVEC, the anti-angiogenic activity of Quinacrine is independent of its pro-apoptotic activity in GMVECs. Quinacrine inhibits glioblastoma angiogenesis and growth in vivo, and acts synergistically with Temozolomide in inhibiting glioblastoma growth in mice. Mechanistically, we found that Quinacrine acts on glioblastoma through inducing oxidative stress, impairing mitochondrial function and activating AMP-activated protein kinase (AMPK). Our work is the first to demonstrate the anti-angiogenic activity of Quinacrine. Our findings highlight Quinacrine as an attractive candidate to support treatment of glioblastoma.

Abstract 3841: SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro

Abstract Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can occur in laminin-111-containing Matrigel and requires the PI3K pathway. However, the membrane protein component and signaling pathways involved in this process are unknown. Methods: In an established in vitro model of VM of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized gene silencing and blocking antibodies to elucidate the signaling pathways involved in this process. RNASeq was used to identify novel transcripts and siRNA was utilized to verify the requirement of candidate RNA/protein in tubular formation. Results: siRNA and antibody blocking of integrin β1, but not β3, prevented VM formation in vitro. Individual silencing of cortactin, TKS5, MMP-2, MMP-9 and MMP-14 affected tubular formation. RNAseq analysis suggested that VM has minimal dependence on de novo transcriptional activity yet reported a strong upregulation of small Integral Membrane Protein 11 (SMIM11A). This result was verified by qPCR and siRNA silencing of SMIM11A prevented VM formation. Discussion: Laminin 111 may interact with integrin β1, and the consequent activation of the PI3K pathway could potentially remodel cytoskeletal proteins and promote the activity of MMPs. We report for the first time a biological role for SMIM11a. This gene is regulated at the RNA level and is required for the formation of tubular structure in vitro. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Javiera Pradenas, Nicole Babbitt, Pamela González, Cristina Bertocchi, Gareth Owen. SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3841.

Effect of Cyclic Uniaxial Mechanical Strain on Endothelial Progenitor Cell Differentiation.

Endothelial progenitor cells (EPCs) have been used as an autologous or allogeneic source in multiple tissue engineering applications. EPCs possess high proliferative and tissue regeneration potential. The effect of shear stress on EPCs has been extensively studied but the role of cyclic mechanical strain on EPCs remains to be understood. In this study, we focused on examining the role of uniaxial cyclic strain on EPCs cultured on three-dimensional (3D) anisotropic composites that mimic healthy and diseased aortic valve tissue matrix compositions. The composites were fabricated by combining centrifugal jet spun fibers with photocrosslinkable gelatin and glycosaminoglycan hydrogels. A custom-designed uniaxial cyclic stretcher was used to provide the necessary cyclic stimulation to the EPC-seeded 3D composites. The samples were cyclically strained at a rate of 1Hz at 15% strain mimicking the physiological condition experienced by aortic valve, with static conditions serving as controls. Cell viability was high in all conditions. Immunostaining revealed reduced endothelial marker (CD31) expression with increased smooth muscle cell marker, SM22α, expression when subjected to cyclic strain. Functional analysis through Matrigel assay agreed with the immunostaining findings with reduced tubular structure formation in strained conditions compared to EPC controls. Additionally, the cells showed reduced acLDL uptake compared to controls which are in alignment with the EPCs undergoing differentiation. Overall, we show that EPCs lose their endothelial progenitor phenotype, and have the potential to be differentiated into mesenchymal-like cells through cyclic mechanical stimulation.

Enhancing the Angiogenic and Proliferative Capacity of Dermal Fibroblasts with Mulberry (Morus alba. L) Root Extract.

Enhancing blood flow and cell proliferation in the hair dermis is critical for treating hair loss. This study was designed to aid the development of alternative and effective solutions to overcome alopecia. Specifically, we examined the effects of Morus alba. L root extract (MARE, which has been used in traditional medicine as a stimulant for hair proliferation) on dermal fibroblasts and other cell types found in the epidermis. We first optimized the concentration of MARE that could be used to treat human dermal fibroblasts (HDFs) without causing cytotoxicity. After optimization, we focused on the effect of MARE on HDFs since these cells secrete paracrine factors related to cell proliferation and angiogenesis that affect hair growth. Conditioned medium (CM) derived from MARE-treated HDFs (MARE HDF-CM) was used to treat human umbilical vein endothelial cells (HUVECs) and hair follicle dermal papilla cells (HFDPCs). Concentrations of MARE up to 20 wt% increased the expression of proliferative and anti-apoptotic genes in HDFs. MARE HDF-CM significantly improved the tubular structure formation and migration capacity of HUVECs. Additionally, MARE HDF-CM treatment upregulated the expression of hair growth-related genes in HFDPCs. CM collected from MARE-treated HDFs promoted the proliferation of HFDPCs and the secretion of angiogenic paracrine factors from these cells. Since it can stimulate the secretion of pro-proliferative and pro-angiogenic paracrine factors from HDFs, MARE has therapeutic potential as a hair loss preventative.

Abstract 2792: Galectin 9 promotes invasion and angiogenesis in vitro and associates with an immune-suppressive microenvironment in gastric cancer patients

Abstract The development of immune checkpoint inhibitors is revolutionizing gastric cancer (GC) therapy. However, the presence of an immune-suppressive tumor microenvironment and redundancy between inhibitory immune checkpoints may be responsible for why a high percentage of patients without clinical benefit. An emerging checkpoint target is Tim-3, a membrane protein that enhances immunosuppression upon binding to its ligand Galectin-9 (Gal-9). Gal-9 is overexpressed in gastrointestinal cancers and expressed in both cancer and stromal cells such as the tumor endothelium. However the role of the Gal-9/Tim-3 pathway in the GC tumor microenvironment is still undeciphered, we hypothesize that this pathway mediates immune escape though enhancement of a immunosuppressive microenvironment. We performed a bioinformatic analysis of stomach adenocarcinoma (STAD) from the TCGA database (TIMER©, Cibersort and Xcell were used). To evaluate if Gal-9 had an impact on cancer cell phenotype we transfected Gal-9 into the gastric cancer cell line AGS and the gastric epithelium cell line GES-1. To assess the potential role in CG tumor microenvironment, Gal-9 was transfected or recombinant Gal-9 (rGal-9) was administrated to Human Umbilical Vein Endothelial Cells (HUVECs). Gal-9 biological effects were assessed by migration, invasion and Matrigel tube formation assay. Western blotting assessed Tim-3 expression and PDL-1 expression. Furthermore, we used lactose to block Gal-9 binding to Tim-3 to determine if the biological effects required receptor engagement. Results from TCGA analysis demonstrated that both Gal-9 and Tim-3 are upregulated in gastric tumors. Tim-3 mRNA is significantly increased in invasive adenocarcinomas. A positive correlation was observed between Gal-9, PDL-1 and Tim-3, which was consistent with in vitro experiments, where increased expression of Gal-9 resulted in increased PDL-1 and Tim-3. In addition, Gal-9 increased cancer cell invasion and promoted HUVEC migration and tubular like structure formation in vitro. Further bioinformatic analysis revealed strong positive correlation with CD8+ T cell, Treg and macrophage/monocyte gene signatures. Tim-3 and Gal-9 were more strongly associated with these gene signatures than PD1 and PDL-1. Interestingly, Gal-9 was associated with T cell dysfunction and Treg markers, suggesting that in GC Gal-9 could promote T cell dysfunction and Treg expansion. Our results suggest an association between the Gal-9/Tim-3 pathway with a more aggressive and increased immune suppressive microenvironment and GC. Funding: Fondecyt 118024, CONICYT-FONDAP 15130011, IMIIP09/16F, FONDECYT 1180173 Citation Format: Charlotte Nicole Hill, Maximiliano Arce Arata, Camille Cabrolier, Noymar Luque, Pamela Gonzalez, Gabriela Maita, Ana Maria Vega-Letter, Patricia Luz Crawford, Marcelo Garrido, Gareth I. Owen. Galectin 9 promotes invasion and angiogenesis in vitro and associates with an immune-suppressive microenvironment in gastric cancer patients [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 2792.

New Insights into the Clinical Implications of Yes-Associated Protein in Lung Cancer: Roles in Drug Resistance, Tumor Immunity, Autophagy, and Organoid Development.

Simple SummaryInnovative advancements in lung cancer treatment have developed over the past decade with the advent of targeted and immune therapies. Yes-associated protein (YAP), an effector of the Hippo pathway, promotes the resistance of these targeted drugs and modulates tumor immunity in lung cancer. YAP is involved in autophagy in lung cancer and plays a prominent role in forming the tubular structure in lung organoids and alveolar differentiation. In this review, we discuss the central roles of YAP in lung cancer and present YAP as a novel target for treating resistance to targeted therapies and immunotherapies in lung cancer.Despite significant innovations in lung cancer treatment, such as targeted therapy and immunotherapy, lung cancer is still the principal cause of cancer-associated death. Novel strategies to overcome drug resistance and inhibit metastasis in cancer are urgently needed. The Hippo pathway and its effector, Yes-associated protein (YAP), play crucial roles in lung development and alveolar differentiation. YAP is known to mediate mechanotransduction, an important process in lung homeostasis and fibrosis. In lung cancer, YAP promotes metastasis and confers resistance against chemotherapeutic drugs and targeted agents. Recent studies revealed that YAP directly controls the expression of programmed death-ligand 1 (PD-L1) and modulates the tumor microenvironment (TME). YAP not only has a profound relationship with autophagy in lung cancer but also controls alveolar differentiation, and is responsible for tubular structure formation in lung organoids. In this review, we discuss the various roles and clinical implications of YAP in lung cancer and propose that targeting YAP can be a promising strategy for treating lung cancer.