Endothelial Cell Tube Formation Research Articles

Targeting the histone methyltransferase SETD7 rescues diabetes-induced impairment of angiogenic response by transcriptional repression of semaphorin 3G

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

Extracellular vesicle-incorporated long noncoding RNA PUNISHER is increased in coronary artery disease and regulates endothelial cell function

Abstract Aims Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. Circulating lncRNAs can be incorporated into extracellular vesicles (EVs). Our aim was to study the expression pattern of circulating EV-incorporated lncRNAs in patients with and without coronary artery disease (CAD). The regulation of the lncRNA PUNISHER was further assessed as an intercellular messenger in endothelial cell (EC) biology. Methods and results Circulating EVs were isolated from the plasma of patients using ultracentrifugation. Electron microscopy, western blot, and NTA were used to determine the size and origin of the EVs. PCR-based human lncRNA array analysis revealed that certain EV-lncRNAs are significantly different in patients with CAD compared to patients without CAD. To validate the lncRNA array results, 60 patients with (n=30) or without (n=30) CAD were prospectively studied. Four atherosclerosis-related lncRNAs (PUNISHER, GAS5, MALAT1, and H19) were quantified in the circulating EVs by using real-time quantitative PCR (RT-qPCR). Among these, PUNISHER (p=0.002) and GAS5 (0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Analysis of RNA degradation revealed that circulating PUNISHER is mainly incorporated within EVs. In vitro, atherosclerotic stimuli consisting of oxLDL or TNF-a treatment caused an upregulation of PUNISHER levels in human coronary artery endothelial cells (HCAEC) and in the corresponding EVs. Labeling of EVs followed by RT-qPCR demonstrated that functional PUNISHER was transported into the recipient ECs, which accelerated cell migration, proliferation, and tube formation. Mechanistically, the RNA-binding protein hnRNPK was identified by an RNA immunoprecipitation (RIP) assay as an interaction partner of PUNISHER, regulating its loading into small EVs. Knockdown of PUNISHER abrogated the EV-mediated effects on EC migration, proliferation, and tube formation. PCR-based gene profiling showed that the expression of VEGF RNA was significantly increased in ECs by treatment with EVs. Protein stability and RNA-immunoprecipitation followed by RT-qPCR analysis indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Knockdown of PUNISHER in EVs abrogated the EV-mediated promotion of VEGFA gene- and protein expression. Conclusion The circulating lncRNA PUNISHER is increased in CAD patients. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.

Colorectal Cancer Cell-Derived Extracellular Vesicles Promote Angiogenesis Through JAK/STAT3/VEGFA Signaling

Background: Angiogenesis plays a crucial role in the growth of colorectal cancer (CRC). Recent studies have identified extracellular vesicles (EVs) in the tumor microenvironment as important mediators of cell-to-cell communication. However, the specific role and mechanisms of CRC-derived EVs in regulating tumor angiogenesis remain to be further investigated. Methods: EVs were isolated from the conditioned medium of the CRC cells using ultracentrifugation. We investigated the effects of HT-29-derived EVs on tumor growth and angiogenesis in a subcutaneous HT-29 CRC tumor model in mice. Additionally, we evaluated the impact of HT-29-derived EVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, bioinformatics analysis was performed to identify relevant signaling pathways, and pathway inhibitors were used to block the activation of these pathways, aiming to elucidate their roles in angiogenesis. Results: We found that HT-29-derived EVs can promote tumor growth and angiogenesis in vivo, as well as significantly enhance the proliferation, migration, and tube formation of HUVECs. Bioinformatics analysis revealed that HT-29-derived EVs may regulate angiogenesis through the JAK/STAT3 signaling pathway. Specifically, we observed that CRC-derived EVs promoted the phosphorylation of STAT3 (p-STAT3) and the expression of VEGFA in the nucleus of HUVECs. Treatment with the STAT3 inhibitor Stattic reduced the nuclear expression of p-STAT3, which impaired its function as a transcription factor, thereby inhibiting VEGFA expression and the pro-angiogenic effects of CRC-derived EVs. Conclusions: EVs derived from CRC cells promote CRC tumor angiogenesis by regulating VEGFA through the JAK/STAT3 pathway in endothelial cells.

CD90's role in vascularization and healing of rib fractures: insights from Dll4/notch regulation.

Vascularization after rib fracture is a crucial physiological process that is essential for the repair and healing of the rib. Studies have shown that CD90 plays a critical role in regulating rib fracture healing, but the underlying mechanism of its role has not been fully elucidated. CD90 adenovirus knockout mice were used to construct a rib injury model. The bone healing was observed by micro-CT. CD31/EMCN immunofluorescence staining was performed on bone tissue to observe the density of H-shaped and L-shaped blood vessels at the site of bone injury. CD31 and EMCN dual-stained single cells from the rib fracture sites were detected by flow cytometry. The periosteal stem cells transfected with CD90 or Notch1 overexpression and silencing vector were co-cultured with osteoblast MC3T3-E1 in osteogenic induction medium. Moreover, bone microvascular endothelial cells were extracted from the rib injury and co-cultured with the periosteal stem cells transfected with CD90. CCK-8 was used to detect cell viability, RT-qPCR and Western blot were used to detect Notch1, Notch2, Notch3, Notch4, CD31, HIF-1α, CD90, RUNX2, OCN and OPN expression. Alkaline phosphatase (ALP) staining and alizarin red staining were used to observe mineralized nodules. Immunofluorescence staining was used to detect the expression of Dll4, Notch, and CD90 in each group of cells. The angiogenesis experiment was conducted to observe cellular vascular formation. Compared with the Adsh-NC group, the bone healing in the Adsh-CD90 group was significantly impaired, with a marked reduction in the number and volume of blood vessels at the rib fracture site, as evidenced by CD31/EMCN immunofluorescence staining, which showed a reduction in the number of H type vessels at the site of bone injury. It was found that CD90 depletion can inhibit the signaling of Dll4/Notch in the rib fracture site. Furthermore, we found that overexpression of Notch1 reverses the impairment of tubule formation in bone microvascular endothelial cells caused by CD90 suppression.r.Dll4 protein reverses the inhibitory effect of CD90 deletion on periosteal stem cells and MC3T3-E1 cell viability and osteogenesis. In the end, we found that overexpression of Notch1 and CD90 can promote angiogenesis of bone microvascular endothelial cells and Notch pathway activation. CD90 can affect vascular formation in mouse rib fractures, and CD90 may be regulated by Dll4/Notch.

Genetically Engineered Filamentous Bacteriophages Displaying TGF-β1 Promote Angiogenesis in 3D Microenvironments

Combined 3D cell culture in vitro assays with microenvironment-mimicking systems are effective for cell-based screening tests of drug and chemical toxicity. Filamentous bacteriophages have diverse applications in material science, drug delivery, tissue engineering, energy, and biosensor development. Specifically, genetically modified bacteriophages have the potential to deliver therapeutic molecules or genes to targeted tumor tissues. The engineered bacteriophages in this study significantly enhanced endothelial cell migration and tube formation within the extracellular matrix (ECM). Compared to TGF-β1 alone and non-modified phages, the presence of TGF-β1 on the bacteriophages demonstrated superior performance as a continuous stimulant in the microenvironment, effectively promoting these angiogenic processes. Assays, including RT-qPCR, ELISA, and fluorescence microscopy, confirmed the expression of angiogenic markers such as CD31, validating the formation of 3D angiogenic structures. Our findings indicate that the TGF-β1 displayed by bacteriophages likely acted as a chemotactic factor, promoting the migration, proliferation, and tube formation of endothelial cells (ECs) within the ECM. Although direct contact between ECs and bacteriophages was not explicitly confirmed, the observed effects strongly suggest that TGF-β1-RGD bacteriophages contributed to the stimulation of angiogenic processes. The formation of angiogenic structures by ECs in the ECM was confirmed as three-dimensional and regulated by the surface treatment of microfluidic channels. These results suggest that biocompatible TGF-β1-displaying bacteriophages could continuously stimulate the microenvironment in vitro for angiogenesis models. Furthermore, we demonstrated that these functionalized bacteriophages have the potential to be utilized as versatile biomaterials in the field of biomedical engineering. Similar strategies could be applied to develop angiogenic matrices for tissue engineering in in vitro assays.

Multifunctional carboxymethyl chitosan/oxidized carboxymethyl cellulose hydrogel loaded with ginsenoside Rg1 and polydopamine nanoparticles for infected diabetic wound healing

Besides bacterial infection, diabetic wounds are often accompanied by local inflammatory response, oxidative stress imbalances, and vascular formation disorders, which are the main reasons for long-term non-healing of diabetic wounds. In order to solve this problem, Ch-OCMC-PDA NPs-Rg1 self-healing hydrogel was constructed by Schiff base reaction. With the addition of PDA NPs and Rg1, Ch-OCMC-PDA NPs-Rg1 hydrogel showed excellent physical properties, like compressive strength of 142 kPa, swelling ratio of 148.91 %, and Rg1 carried in the hydrogel could achieve a slow release of 90.59 % within 48 h. What's more, PDA NPs endowed it with highly efficient photothermal antibacterial properties. In addition to excellent biocompatibility, Ch-OCMC-PDA NPs-Rg1 hydrogel could effectively clear intracellular reactive oxygen species, promote macrophages M2 transformation, and facilitate human umbilical vein endothelial cells migration and tube formation. In vivo experiments exhibited that Ch-OCMC-PDA NPs-Rg1 hydrogel could reduce wound inflammation, stimulate early angiogenesis, promote collagen deposition, and shorten the healing process of diabetic infected wounds, and the wound healing rate was significantly increased compared with other groups, reaching 98.41 ± 0.31 %. In summary, the multi-functional dynamic Ch-OCMC-PDA NPs-Rg1 hydrogel provides a new possibility for the treatment of diabetic infection wounds.

Differential Effects of Four Canonical Notch-Activating Ligands on c-Kit+ Cardiac Progenitor Cells.

Notch signaling, an important signaling pathway in cardiac development, has been shown to mediate the reparative functions of c-kit+ progenitor cells (CPCs). However, it is unclear how each of the four canonical Notch-activating ligands affects intracellular processes in c-kit+ cells when used as an external stimulus. Neonatal c-kit+ CPCs were stimulated using four different chimeric Notch-activating ligands tethered to Dynabeads, and the resulting changes were assessed using TaqMan gene expression arrays, with subsequent analysis by principal component analysis (PCA). Additionally, functional outcomes were measured using an endothelial cell tube formation assay and MSC migration assay to assess the paracrine capacity to stimulate new vessel formation and recruit other reparative cell types to the site of injury. Gene expression data showed that stimulation with Jagged-1 is associated with the greatest pro-angiogenic gene response, including the expression of VEGF and basement membrane proteins, while the other canonical ligands, Jagged-2, Dll-1, and Dll-4, are more associated with regulatory and epigenetic changes. The functional assay showed differential responses to the four ligands in terms of angiogenesis, while none of the ligands produced a robust change in migration. These data demonstrate how the four Notch-activating ligands differentially regulate CPC gene expression and function.

Experimental study on repairing rat abdominal wall defect with chitosan hydrogel/polypropylene mesh composite

To investigate the improvement effects and mechanisms of composite chitosan (CS) hydrogel on traditional polypropylene (PP) mesh for repairing abdominal wall defects. CS hydrogel was prepared via physical cross-linking and then combined with PP mesh to create a CS hydrogel/PP mesh composite. The internal structure and hydrophilicity of the composite were characterized using macroscopic observation, upright metallographic microscope, scanning electron microscopy, and water contact angle measurements. The performance of the composite (experimental group) in resisting cell adhesion and supporting cell infiltration was assessed through fibroblast (NIH-3T3) infiltration experiments and human umbilical vein endothelial cells (HUVECs) tube formation assays, and simple cells were used as control group. Finally, a bilateral abdominal wall defect model (1.5 cm×1.0 cm) was established in 18 Sprague Dawley rats aged 8-10 weeks, with the composite used on one side (experimental group) and PP mesh on the other side (control group). The effects on promoting wound healing, preventing adhesion, angiogenesis, and anti-inflammation were investigated through macroscopic observation, histological staining (HE and Masson staining), and immunohistochemical staining (CD31, CD68). The composite appeared as a pale yellow, transparent solid with a thickness of 2-3 mm, with the PP mesh securely encapsulated within the hydrogel. Scanning electron microscopy revealed that the hydrogel contained interconnected pores measuring 100-300 μm, forming a porous structure. Contact angle measurements indicated that CS hydrogel exhibited good hydrophilicity, while PP mesh was highly hydrophobic. In vitro cell culture experiments showed that DAPI staining indicated fewer positive cells in the experimental group after 1 day of culture, while the cells in control group covered the entire well plate. After 3 days of culture, the cells in experimental group were spherical and displayed uneven fluorescence, suggesting that the material could reduce cell adhesion while supporting cell infiltration. HUVECs tube formation experiments demonstrated an increase in cell numbers in experimental group with a trend towards tube formation, while cells in control group were sparsely distributed and showed no migration. In the rat abdominal wall defect repair experiment, results showed that after 1 week post-surgery, the experimental group had tissue and blood vessels infiltrating, and by 4 weeks, the integrity was well restored with significant regeneration of muscle and blood vessels, while the control group exhibited adhesions and incomplete healing. HE staining results indicated weaker cell infiltration in the experimental group, with cell density significantly higher than that of the control group at 2 and 4 weeks post-surgery ( P<0.05). Masson staining revealed that collagen fibers in the experimental group were arranged neatly, with significantly increased collagen content at 2 weeks post-surgery ( P<0.05), while collagen content was similar in both groups at 4 weeks ( P>0.05). Immunohistochemical staining showed that CD31-positive cells were evenly distributed between muscle layers in the experimental group, whereas the control group exhibited notable defects. At 2 weeks after operation, the CD31-positive cell ratio was significantly higher than that in the control group ( P<0.05); at 2 and 4 weeks after operation, the CD68-positive cell ratio in the experimental group was significantly lower than that in the control group ( P<0.05). CS hydrogel has a positive effect on preventing adhesions and promoting wound healing, exhibiting anti-inflammatory and pro-angiogenic properties during the healing process. This provides a promising strategy to address challenges related to abdominal adhesions and reconstruction.

Exosomes from hypoxic urine-derived stem cells facilitate healing of diabetic wound by targeting SERPINE1 through miR-486-5p

Vascular pathologies and injuries are important factors for the delayed wound healing in diabetes. Previous studies have demonstrated that hypoxic environments could induce formation of new blood vessels by regulating intercellular communication and cellular behaviors. In this study, we have enhanced the angiogenic potential of exosomes by subjecting urine-derived stem cells (USCs) to hypoxic preconditioning. To prolong the retention of exosomes at the wound site, we have also engineered a novel dECM hydrogel termed SISMA, which was modified from porcine small intestinal submucosa (SIS). For its rapid and controllable gelation kinetics, excellent biocompatibility, and exosome release capability, the SISMA hydrogel has proven to be a reliable delivery vehicle for exosomes. The hypoxia-induced exosomes-loaded hydrogel has promoted endothelial cell proliferation, migration, and tube formation. More importantly, as evidenced by significant in vivo vascular regeneration in the early stages post-injury, it has facilitated tissue repair. This may because miR-486–5p in H-exo inhibit SERPINE1 activity in endothelial cell. Additionally, miRNA sequencing analysis suggested that the underlying mechanism for enhanced angiogenesis may be associated with the activation of classical HIF-1α signaling pathway. In summary, our study has presented a novel non-invasive, cell-free therapeutic approach for accelerating diabetes wound healing and development of a practical and efficient exosomes delivery platform.

Cancer-associated Fibroblasts-derived Exosomes with HOXD11 Overexpression Promote Ovarian Cancer Cell Angiogenesis Via FN1.

Cancer-associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote metastasis in patients with ovarian cancer (OC). Here, we try to further understand the mechanism by which CAFs-derived exosomes (CAFs-Exo) promoted angiogenesis in OC. We intersected differentially expressed genes in OC cells after CAFs-Exo treatment in the GSE147610 dataset with a list of transcription factors to identify homeobox protein hox-D11 (HOXD11) as a possible cargo of CAFs-Exo. HOXD11 encapsulated by CAFs-Exo enhanced colony formation, migration, and invasion of OC cells. HOXD11 bound to the promoter of fibronectin (FN1) and promoted its transcription. HOXD11 knockdown from CAFs-Exo significantly repressed the VEGF and CD31 protein expression and tube formation, viability, and migration of human umbilical vein endothelial cells (HUVEC) and slowed angiogenesis and tumor growth in mice. Furthermore, we found that overexpression of FN1 increased the expression of angiogenic factors and activity of HUVEC in the presence of HOXD11 knockdown. These results verify the significant contribution of CAFs-Exo to angiogenesis in OC, which could be partially due to the promotion of FN1 mediated by HOXD11.

Bioactive MXene hydrogel promotes structural and functional regeneration of skeletal muscle through improving autophagy and muscle innervation

Complete skeletal muscle regeneration after traumatic injuries remains a challenge due to impaired regenerative capability and dysregulated microenvironments. Autophagy plays a crucial role in the muscle regeneration process by regulating myogenic and non-myogenic cells. Herein, we report a bioactive MXene hydrogel (FPGM) capable of upregulating autophagy and increasing muscle innervation to restore skeletal muscle structure and function. FPGM possessed excellent electrical conductivity, tissue adhesive ability and antioxidation, which could eliminate excess reactive oxygen species to reduce oxidative stress and decrease the secretion of pro-inflammatory cytokine. FPGM upregulated the autophagy level of myoblasts and promoted the migration and tube formation of endothelial cells as well as myogenic differentiation with negligible toxicity. FPGM accelerated muscle fiber formation and skeletal muscle regeneration by improving autophagy, which could regulate microenvironment through raising M2 macrophages to alleviate excessive inflammation, facilitating angiogenesis and decreasing fibrous scar tissue formation in vivo. Importantly, FPGM could efficiently restore muscle function by improving muscle innervation, tibialis anterior compound muscle action potential amplitude and neuromuscular conduction. This work demonstrates that bioactive MXene hydrogel should be a promising candidate for complete skeletal muscle regeneration.

Integrated miRNA sequencing and experimental validation Unveil that low-level laser enhances vascular endothelial cell proliferation, migration, and lumen formation via miR-90/VEGFA

The hydroxyapatite orbital implantation is widely used to treat orbital malformation, but delayed postoperative angiogenesis can hinder conjunctival wound healing, potentially leading to implant exposure and prolapse. Low-intensity laser therapy (LLLT) is recognized for its ability to promote tissue regeneration, reduce inflammation, and alleviate pain. This study aims to explore the specific mechanism of miRNAs-VEGFA pathway regulation in early vascularization after orbital implant placement induced by LLLT. A hydroxyapatite orbital implant model was established and treated with LLLT. Vascular tissues surrounding the ocular prosthesis were extracted for high-throughput sequencing to identify differentially expressed miRNAs. miRNAs predicted to bind with VEGFA were selected for validation. GO and KEGG analyses were performed to reveal the functional enrichment of target genes regulated by these miRNAs. Dual luciferase assay, qRT-PCR, and Western blotting were used to verify the targeting relationship between miR-90 and VEGFA. The effects of miR-90 on rabbit microvascular endothelial cell function were assessed through CCK-8 assay, scratch test, and tube formation assay. High-throughput sequencing revealed 32 differentially expressed miRNAs, with 8 upregulated and 24 downregulated. miR-90 was predicted to have a high binding score and expression abundance with VEGFA and was confirmed to regulate VEGFA expression. In vitro functional tests showed that miR-90 inhibited rabbit microvascular endothelial cell proliferation, migration, and tube formation. This study is the first to demonstrate that LLLT regulates ocular prosthesis angiogenesis via the miR-90/VEGFA pathway, providing a new target for treating vascular-dependent diseases.

A multi-purpose dressing based on resveratrol-loaded ionic liquids/gelatin methacryloyl hydrogel for enhancing diabetic wound healing

Diabetic wound (DW) is a multifaceted challenge, characterized by persistent bacterial infections and compromised angiogenesis. To address these issues and enhance DW healing, we developed a novel strategy using a photo-crosslinked hydrogel system composed of ionic liquids (ILs) and gelatin methacryloyl (GelMA) loaded with resveratrol (Res). The ILs/GelMA hydrogel was fabricated via a simple photo-crosslinking process, resulting in desirable mechanical properties, biocompatibility, and controlled release kinetics. Res was incorporated into the hydrogel matrix (ILs/GelMA@Res) to ensure sustained release, facilitating angiogenesis and accelerating wound healing. In vitro studies demonstrated that the ILs/GelMA@Res hydrogel exhibited potent antibacterial activity against Staphylococcus aureus and Escherichia coli, inhibiting bacterial growth and biofilm formation. Furthermore, the sustained release of Res from the hydrogel promoted angiogenesis by activating the PI3K/AKT signaling pathways associated with VEGF and FGF, enhancing endothelial cell proliferation, migration, and tube formation. In a DW mice model, the ILs/GelMA@Res hydrogel demonstrated accelerated wound closure, reduced inflammation, and robust neovascularization. This multifunctional hydrogel-based delivery system holds considerable potential for clinical translation, offering a safe and effective treatment modality for diabetic patients with chronic wounds.

Methyl Gallate Suppresses Canine Mammary Gland Tumors by Inducing Apoptosis and Anti-angiogenesis.

Methyl gallate (MG), a plant phenolic compound, has known anticancer properties. However, its effects on canine mammary gland tumors (CMTs) are unclear. This study evaluated the impact of MG on cell viability, migration, and apoptosis in two CMT cell lines. CMT-U27 and CF41.mg cells were used. In vitro experiments included MTT and scratch assays, Annexin-V/propidium iodide double staining, immunocytochemistry, and western blot analyses. An in vivo CMT xenograft mouse model was also used to observe the effects of MG on tumor growth and vasculature. Immunohistochemistry was performed to analyze vessel density and apoptosis in tumor tissues. Cell migration and tube formation assays with canine aortic endothelial cells assessed the anti-angiogenic effects of MG. Data showed a significant decrease in cell viability and migration in both CMT cell lines after 24 h exposure to various MG concentrations. MG treatment induced dose-dependent apoptotic cell death and elevated cleaved caspase-3 expression. In vivo experiments confirmed tumor growth suppression 21 days post-treatment with 40 mg/kg MG. Tumor tissues displayed increased cleaved caspase-3 and reduced vessel density. MG also inhibited cell migration and disrupted tube formation in canine endothelial cells. MG has potential as an anticancer drug for CMTs by promoting apoptotic cell death and reducing angiogenesis, highlighting its therapeutic promise.

Engineering Three-Dimensional Spheroid Culture for Enrichment of Proangiogenic miRNAs in Umbilical Cord Mesenchymal Stem Cells and Promotion of Angiogenesis.

In the field of regenerative medicine, umbilical cord-derived mesenchymal stem cells (UC-MSCs) have a plausible potential. However, traditional two-dimensional (2D) culture systems remain limited in replicating the complex in vivo microenvironment. Thus, three-dimensional (3D) cultures offer a more physiologically relevant model. This study explored the impact of 3D culture conditions on the UC-MSC secretome and its ability to promote angiogenesis, both in vitro and in vivo. In this study, using two distinct methods, we successfully cultured UC-MSCs: in a monolayer (2D-UC-MSCs) and as spheroids formed in U-shaped 96-well plates (3D-UC-MSCs). The presence and expression of proangiogenic miRNAs in the conditioned media (CM) of these cultures were investigated, and differential expression patterns were explored. Particularly, the CM of 3D-UC-MSCs revealed significantly higher levels of miR-21-5p, miR-126-5p, and miR-130a-3p compared to 2D-UC-MSCs. Moreover, the CM from 3D-UC-MSCs revealed a higher effect on endothelial cell proliferation, migration, and tube formation than did the CM from 2D-UC-MSCs, indicating their proangiogenic potential. In an in vivo Matrigel plug mouse model, 3D-UC-MSCs (cells) stimulated greater vascular formation compared to 2D-UC-MSCs (cells). 3D culture of UC-MSCs' secretome improves the promotion of angiogenesis.

Biomimetic Multichannel Silk Nerve Conduits With Multicellular Spatiotemporal Distributions for Spinal Cord Injury Repair.

Bioengineered nerve conduits have shown great promise for spinal cord injury (SCI) repair, while their practical values are limited by poor regenerative efficacy and lack of multi-level structural design. Here, inspired by the ingenious anatomy of natural spinal cords, a biomimetic multichannel silk nerve conduit (namely BNC@MSCs/SCs) with multicellular spatiotemporal distributions for effective SCI repair is presented. The biomimetic silk nerve conduit (BNC) with hierarchical channels and aligned pore structures is prepared via a modified directional freeze-casting strategy. Such hierarchical structures provide appropriate space for the mesenchymal stem cells (MSCs) and Schwann cells (SCs) settled in specific channels, which contributes to the generation of BNC@MSCs/SCs resembling the cellular spatiotemporal distributions of natural spinal cords. The in vitro results reveal the facilitated SC migration and MSC differentiation in such BNC@MSCs/SCs multicellular system, which further promotes the tube formation and cell migration of endothelial cells as well as M2 polarization of macrophages. Moreover, BNC@MSCs/SCs can effectively promote the tissue repair and function recovery in SCI rats by attenuating glial scar formation while promoting neuron regeneration and myelin sheath reconstruction. Thus, it is believed that the biomimetic multichannel silk nerve conduits with multicellular spatiotemporal distributions are valuable for SCI repair and other neural tissue regeneration.

Heterogeneous porous hypoxia-mimicking scaffolds propel urethral reconstruction by promoting angiogenesis and regulating inflammation

The nasty urine microenvironment (UME) impedes neourethral regeneration by inhibiting angiogenesis and inducing an excessive inflammatory response. Cellular adaptation to hypoxia improves regeneration in numerous tissues. In this study, heterogeneous porous hypoxia-mimicking scaffolds were fabricated for urethral reconstruction via promoting angiogenesis and modulating the inflammatory response based on sustained release of dimethyloxalylglycine (DMOG) to promote HIF-1α stabilization. Such scaffolds exhibit a two-layered structure: a dense layer composed of electrospun poly (l-lactic acid) (PLLA) nanofibrous mats and a loose layer composed of a porous gelatin matrix incorporated with DMOG-loaded mesoporous silica nanoparticles (DMSNs) and coated with poly(glycerol sebacate) (PGS). The modification of PGS could significantly increase rupture elongation, making the composite scaffolds more suitable for urethral tissue regeneration. Additionally, sustained release of DMOG from the scaffold facilitates proliferation, migration, tube formation, and angiogenetic gene expression in human umbilical vein endothelial cells (HUVECs), as well as stimulates M2 macrophage polarization and its regulation of HUVECs migration and smooth muscle cell (SMCs) contractile phenotype. These effects were downstream of the stabilization of HIF-1α in HUVECs and macrophages under hypoxia-mimicking conditions. Furthermore, the scaffold achieved better urethral reconstruction in a rabbit urethral stricture model, including an unobstructed urethra with a larger urethral diameter, increased regeneration of urothelial cells, SMCs, and neovascularization. Our results indicate that heterogeneous porous hypoxia-mimicking scaffolds could promote urethral reconstruction via facilitating angiogenesis and modulating inflammatory response.

Bioactive Nb2C MXene-Functionalized Hydrogel with Microenvironment Remodeling and Enhanced Neurogenesis to Promote Skeletal Muscle Regeneration and Functional Restoration.

The complete structure-functional repair of volumetric muscle loss (VML) remains a giant challenge and biomedical hydrogels to remodel microenvironment and enhance neurogenesis have appeared to be a promising direction. However, the current hydrogels for VML repair hardly achieve these two goals simultaneously due to their insufficient functionality and the challenge in high-cost of bioactive factors. In this study, a facile strategy using Nb2C MXene-functionalized hydrogel (OPTN) as a bioactive scaffold is proposed to promote VML repair with skeletal muscle regeneration and functional restoration. In vitro experiments show that OPTN scaffold can effectively scavenge reactive oxygen species (ROS), guide macrophages polarization toward M2 phenotype, and resist bacterial infection, providing a favorable microenvironment for myoblasts proliferation as well as the endothelial cells proliferation, migration, and tube formation. More importantly, OPTN scaffold with electroactive feature remarkably boosts myoblasts differentiation and mesenchymal stem cells neural differentiation. Animal experiments further confirm that OPTN scaffold can achieve a prominent structure-functional VML repair by attenuating ROS levels, alleviating inflammation, reducing fibrosis, and facilitating angiogenesis, newborn myotube formation, and neurogenesis. Collectively, this study provides a highly promising and effective strategy for the structure-functional VML repair through designing bioactive multifunctional hydrogel with microenvironment remodeling and enhanced neurogenesis.

FHL2 expression by cancer-associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma.

Cancer-associated fibroblasts (CAFs) contribute to the progression of lung cancer. Four and a half LIM domain protein-2 (FHL2) is a component of focal adhesion structures. We analyzed the function of FHL2 expressed by CAFs in lung adenocarcinoma. Expression of FHL2 in fibroblast subtypes was investigated using database of single-cell RNA-sequencing of lung cancer tissue. The role of FHL2 in the proliferation and migration of CAFs was assessed. The effects of FHL2 knockout on the migration and invasion of human lung adenocarcinoma cells and tube formation of endothelial cells induced by CAF-conditioned medium (CM) were evaluated. The effect of FHL2 knockout in CAFs on metastasis was determined using a murine orthotopic lung cancer model. The prognostic significance of stromal FHL2 was assessed by immunohistochemistry in human adenocarcinoma specimens. FHL2 is highly expressed in myofibroblasts in cancer tissue. TGF-β1 upregulated FHL2 expression in CAFs and FHL2 knockdown attenuated CAF proliferation. FHL2 knockout reduced CAF induced migration of A110L and H23 human lung adenocarcinoma cell lines, and the induction of tube formation of endothelial cells. FHL2 knockout reduced CAF-induced metastasis of lung adenocarcinomas in an orthotopic model in vivo. The concentration of Osteopontin (OPN) in CM from CAF was downregulated by FHL2 knockout. siRNA silencing and antibody blocking of OPN reduced the pro-migratory effect of CM from CAF on lung cancer cells. In resected lung adenocarcinoma specimens, positive stromal FHL2 expression was significantly associated with higher microvascular density and worse prognosis. In conclusion, FHL2 expression by CAFs enhances the progression of lung adenocarcinoma by promoting angiogenesis and metastasis.

Polydatin accelerates osteoporotic bone repair by inducing the osteogenesis-angiogenesis coupling of bone marrow mesenchymal stem cells via the PI3K/AKT/GSK-3β/β-catenin pathway.

Polydatin (POL), a natural stilbenoid, has multiple pharmacological activities. However, its effect on osteoporotic bone defect has not yet been examined. This study was designed to explore the unknown role of POL on osteoporotic bone repair. The effect of POL on osteogenesis and angiogenesis were investigated firstly. Then a series of angiogenesis-related assays were carried out to explore the relationship between osteogenesis and angiogenesis of POL, and the underlying mechanism was further explored. Whereafter, ovariectomy-induced osteoporosis rats with bone defect were treated with POL or placebo, the imageological and histological examinations were conducted to assess the effect of POL on osteoporotic bone repair. The moderate concentrations (1μM and 10μM) of POL enhanced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) and elevated the expression of angiogenic-specific markers. Further research found that POL induced human umbilical vein endothelial cells migration and tube formation through the osteogenesis-angiogenesis coupling of BMSCs, and the POL-induced osteogenesis-angiogenesis coupling was reversed after co-cultured with LY294002, Mechanistically, this was conducted via activating PI3K/AKT/GSK-3β/β-catenin pathway. After that, using osteoporotic bone defect rat model, we observed that POL facilitated osteoporotic bone repair through enhancing osteogenesis and CD31hiEMCNhi type H-positive vessels formation via the PI3K/AKT/GSK-3β/β-catenin pathway. The data above indicated that POL could accelerate osteoporotic bone repair by inducing the osteogenesis-angiogenesis coupling of BMSCs via the PI3K/AKT/GSK-3β/β-catenin pathway, which provided new insight and strategy for osteoporotic bone repair.