Endothelial Cell Tube Formation Research Articles

MiR-25-3p regulates pulmonary arteriovenous malformation after Glenn procedure in patients with univentricular heart via the PHLPP2-HIF-1α axis

The detailed mechanism of pulmonary arteriovenous malformations after Glenn surgery (G-PAVMs) in cyanotic congenital heart disease (CHD) remains unclear. Microarray in situ hybridization was performed to assess the miRNA (miRNA) profiles of serum from pediatric patients (0–6 years of age) with G-PAVMs and after the Fontan procedure without G-PAVMs. In addition, we investigated the tube formation, migration, and proliferation of human lung microvascular endothelial cells (HMVEC-L) transfected with miR-25-3p mimic, miR-25-3p inhibitor, or PHLPP2 small interfering RNA, and examined HIF-1α/VEGF-A signaling after hypoxic stimulation. Serum miRNAs that showed ≥ 2-fold higher levels in patients with G-PAVMs than in other patients were selected. MiR-25-3p was significantly upregulated in the pulmonary artery sera of the post-Glenn group than in the post-Fontan group. We identified PHLPP2 as a direct target of miR-25-3p. PHLPP2 expression was significantly decreased in HMVEC-L transfected with miR-25-3p mimic compared to the control cells. HIF-1α and VEGF-A expression levels were increased in HMVEC-L transfected with miR-25-3p mimic compared to the control cells in a PHLPP2/Akt/mTOR signaling-dependent manner after hypoxic stimulation. MiR-25-3p promoted HMVEC-L angiogenesis, proliferation, and migration under hypoxic conditions. MiR-25-3p in the pulmonary arteries may contribute to G-PAVM development.

Porcine pericardial decellularized matrix bilayer patch containing adipose stem cell-derived exosomes for the treatment of diabetic wounds.

Chronic hard-to-heal wounds pose a significant threat to patients' health and quality of life, and their clinical management remains a challenge. Adipose-derived stem cell exosomes (ADSC-exos) have shown promising results in promoting diabetic wound healing. However, effectively enhancing the retention of exosomes in wounds for treatment remains a key issue that needs to be addressed. There is a pressing need to develop new materials or methods to improve the bioavailability of exosomes. Porcine pericardium, an extracellular matrix-rich tissue, is easily obtainable and widely available. Decellularized porcine pericardium removes cellular components while retaining an extracellular matrix that supports cellular growth, making it an ideal raw material for preparing wound dressings. In this study, we developed porcine pericardial decellularized matrix bilayer patches loaded with ADSC-exos, which were transplanted into diabetic mouse skin wounds. Histological and immunohistochemical analyses revealed that these bilayer matrix patches accelerate wound healing by promoting granulation tissue formation, re-epithelialization, stimulating vascularization, and enhancing collagen production. In terms of the underlying biological mechanism, we found that decellularized extracellular matrix bilayer patches loaded with ADSC-exos enhanced the proliferation and migration of human dermal fibroblasts (HDFs) and HaCaT cells in vitro, and promoted tube formation in human umbilical vein endothelial cells (HUVECs). This research demonstrated that the porcine pericardial decellularized matrix is well-suited for exosome delivery and that these bilayer patches hold great potential in promoting diabetic wound healing, providing evidence to support the future clinical application of ADSC-exos.

Interleukin 29 is a novel antiangiogenic factor in angiogenesis.

Angiogenesis is tightly controlled by growth factors and cytokines in pathophysiological settings. Despite the importance of Interleukin 29 (IL-29), a newly identified cytokine of type III interferon family, its role in angiogenesis remains unknown. We aimed to elucidate IL-29's impact on angiogenesis under both and physiological and pathological conditions. We employed various assays to evaluate IL-29's effect on proliferation, apoptosis, migration and tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. IL-29's angiogenic effect was assessed using mouse aortic rings ex vivo, and oxygen-induced retinopathy (OIR) mouse model in vivo. Signaling pathways possibly involved in IL-29-induced angiogenesis were investigated by Western blot. Finally, IL-29's impact on tube formation was blocked by inhibiting IL-29/interleukin 10 receptor 2 (IL-10R2) binding. IL-29 treatment inhibited endothelial cell migration, tube formation and vessel sprouting, without affecting proliferation or apoptosis. Notably, IL-29 (100ng/ml) attenuated vessel growth in pathological angiogenesis in OIR mice, accompanied by decreased expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). Mechanistically, IL-29 activated Stat3 signaling pathway, and blocking IL-29/IL-10R2 binding remarkably reversed IL-29's anti-angiogenic effect on tube formation. Our findings demonstrated that IL-29, at a relative low concentration, modulates angiogenesis in both physiological and pathological contexts. Targeting IL-29 or its receptor IL-10R2 offers a promising strategy for angiogenesis regulation in various conditions.

MiR-5586-5p Suppresses Hypoxia-induced Angiogenesis Through Multiple Targeting of HIF-1α, HBEGF and ADAM17 in Breast Cancer.

Hypoxia-inducible factor-1 alpha (HIF-1α) plays a key role in the cellular response to hypoxia, which plays a crucial role in the induction of abnormal angiogenesis and metastasis. Understanding the mechanism for the regulation of angiogenesis by HIF-1α-regulating miRNA will contribute to developing the strategy to prevent metastasis. We conducted a functional screening for HIF-1α-inhibiting miRNAs by evaluating the effects of miRNA mimics on HIF-1α expression and identified miR-5586-5p as an angiogenesis inhibitor through a mechanistic study. Angiogenic activity was assessed by tube formation assays using HUVEC cells exposed to conditioned media from miRNA-transfected breast cancer cells. In vivo activity of miR-5586-5p was examined through intratumoral injection of miRNA in orthotopic xenograft mice established by injecting MDA-MB-231 cells into the mammary fat pads of BALB/c nu/nu mice. The expression of the critical proangiogenic factors vascular endothelial growth factor A (VEGFA) and angiopoietin-like protein 4 (ANGPTL4) was inhibited by miR-5586-5p. Migration and tube formation of human umbilical vein endothelial cells were reduced in the conditioned medium prepared from miR-5586-5p-transfected cells. miR-5586-5p also suppressed the expression of heparin-binding EGF-like growth factor (HBEGF) and a disintegrin and metalloprotease 17 (ADAM17), which play a role in hypoxic signaling to induce the expression of VEGFA and ANGPTL4. HIF-1α, HBEGF, and ADAM17 were verified as the direct targets of miR-5586-5p responsible for the angiogenesis-suppressing function of miR-5586-5p. Expression levels of miR-5586-5p were lower in tumor tissues than in neighboring normal tissues of breast cancer patients. The expression of miR-5586-5p was inversely correlated to those of HIF-1α, HBEGF, ADAM17, VEGFA, and ANGPTL4. Angiogenesis and subsequent tumor growth were suppressed by intratumoral injection of miR-5586-5p in orthotopic MDA-MB-231 xenografts in mice. A potent tumor-suppressive function of miR-5586-5p applicable for the development of a novel cancer treatment strategy is herein described.

Extracellular vesicle-associated miR-ERIA exerts the anti-angiogenic effect of macrophages in diabetic wound healing.

Many cell types are involved in the regulation of cutaneous wound healing in diabetes. Clarifying the mechanism of cell-cell interactions is important for identifying therapeutic targets for diabetic cutaneous ulcers. The function of vascular endothelial cells in the cutaneous microenvironment is critical, and a decrease in their biological function leads directly to refractory wound healing. In this study, we aimed to study the interactions of macrophages with vascular endothelial cells and elucidate the mechanism of diabetic wound angiogenesis suppression. We found that macrophages polarized to the M1 type, inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs) by secreting extracellular vesicles after treatment with advanced glycation end products (AGEs-EVs), and contributed to wound angiogenesis and delayed wound healing in vivo. Mechanistically, we identified a novel miRNA enriched in AGEs-EVs, namely miR-ERIA, that suppress the biological function of HUVECs by targeting helicase with zinc finger 2 (HELZ2), and in vivo experiments showed that miR-ERIA suppression could promote wound angiogenesis and thus accelerate wound healing in diabetes. We found that miR-ERIA regulates diabetic wound angiogenesis by targeting HELZ2, suggesting a potential therapeutic target for diabetic foot ulcers.

Gas-propelled anti-hair follicle aging microneedle patch for the treatment of androgenetic alopecia.

Existing treatments for androgenetic alopecia (AGA) are unsatisfactory, owing to the two major reasons: (1) Oxidative stress and vascularization deficiency in the perifollicular microenvironment provoke the premature senescence of hair follicles, limiting the transformation of hair growth cycle from the telogen to the anagen phase; (2) The amount of drug delivered to the perifollicular region located in the deep dermis is very limited for passive drug delivery systems. Herein, we developed a gas-propelled microneedle patch integrated with ferrum-chelated puerarin/quercetin nanoparticles (PQFN) to increase drug accumulation in hair follicles and reshape the perifollicular microenvironment for improved hair-regenerating effects. PQFN can rejuvenate testosterone (Tes)-induced senescence of dermal papilla cells by scavenging ROS, restoring mitochondrial function, regulating signaling pathways related to hair regeneration, and upregulating hair growth-promoting genes. PQFN more efficiently promoted endothelial cell proliferation, migration, and tube formation than ferrum-chelated quercetin nanoparticles (QFN) because of puerarin's proangiogenic effects. Compared with passive MNs, gas-propelled MNs promoted drug diffusion and permeation into deeper skin layers, resulting in significantly higher drug accumulation in hair follicles. Pharmacodynamic studies on an AGA mouse model further showed that PQFN-loaded active MNs achieved higher hair coverage by alleviating oxidative stress, promoting angiogenesis, and rejuvenating senescent cells. Therefore, this study presents a novel "anti-hair follicle aging" treatment strategy for AGA.

Nephronectin Is Required for Vascularization in Zebrafish and Sufficient to Promote Mammalian Vessel-Like Structures in Hydrogels for Tissue Engineering.

Organs and tissues need to be vascularized during development. Similarly, vascularization is required to engineer thick tissues. How vessels are formed during organogenesis is not fully understood, and vascularization of engineered tissues remains a significant challenge. Here, we show that the extracellular matrix protein nephronectin is required for vascularization during zebrafish development as well as adult fin regeneration and is sufficient to promote mammalian vessel formation and maturation. Nephronectin a morphants and mutants exhibit diminished axial vein sprouting and posterior intersegmental vessel growth. Notably, the angiogenesis-associated integrins itgav and itgb3.1 are coexpressed with nephronectin a in the region of the caudal vein plexus and posterior somites; nephronectin binds to integrin alpha-V/integrin beta-3.1 (ITGAV/ITGB3.1), and itgav morphants phenocopy nephronectin a mutants. In addition, nephronectin a mutants showed decreased vessel maturation compared with wild-type siblings during caudal fin regeneration in adult zebrafish. Moreover, nephronectin promotes mammalian endothelial cell migration and tube formation in 2D and 3-dimensional invitro tissue culture. Further, nephronectin enhances vascular endothelial growth factor-induced periaortic vascular capillary interconnectivity, vessel diameter, and vessel stability. Collectively, our results identify nephronectin as a proangiogenic factor during embryonic development, which can be used to improve the vascularization of engineered tissues.

Development of a recombinant Ang1 variant with enhanced Tie2 binding and its application to attenuate sepsis in mice.

The angiopoietin (Ang)-Tie axis, critical for endothelial cell function and vascular development, is a promising therapeutic target for treating vascular disorders and inflammatory conditions like sepsis. This study aimed to enhance the binding affinity of recombinant Ang1 variants to the Tie2 and explore their therapeutic potential. Structural insights from the Ang1-Tie2 complex enabled the identification of key residues within the Ang1 receptor binding domain (RBD) critical for Tie2 interaction. Molecular dynamics simulations revealed that Met436Arg (M436R) and Ala451Asp (A451D) could improve Ang1's Tie2 binding affinity. One variant, Ang1-RBDA451D, demonstrated a 100-fold increase compared to the wild type. Cellular assays revealed that Ang1A451D enhanced Tie2 phosphorylation, promoting endothelial cell migration and tube formation. In vivo, this variant effectively reduced inflammatory cytokines and attenuated organ damage in septic mice. These findings highlight Ang1A451D as a promising therapeutic candidate for vascular diseases, offering notable clinical potential for mitigating sepsis-related vascular dysfunction.

The effect of crocin on the proliferation, inflammation, drug synergism, and angiogenesis in breast cancer

Radiation, chemotherapy, and surgery are common treatments for breast cancer. These approaches, however, could only improve the odds of surviving. Using herbal medicines like crocin is now necessary to improve cancer treatment, particularly for breast cancer. According to research, crocin inhibits the growth of cancerous cells while having no effect on the development of healthy cells. By eating this substance, the side effects of cancer chemotherapy are also reduced. Thus, this review delves into the effects of crocin on breast cancer, encompassing anti-angiogenesis, drug synergism, proliferation, and inflammation. Crocin decreases the expression of CD34 in tumor tissues, leading to anti-angiogenesis. Furthermore, the anti-angiogenic effect of crocin can hinder vascular endothelial cell proliferation, migration, and tubule formation in vitro, as well as tumor cell proliferation and reduction in microvascular density in vivo. Also, crocin increases the sensitivity of PTX in breast cancer cell lines.

Exosomes derived from highly scalable and regenerative human progenitor cells promote functional improvement in a rat model of ischemic stroke.

Globally, there are 15 million stroke patients each year who have significant neurological deficits. Today, there are no treatments that directly address these deficits. With demographics shifting to an older population, the problem is worsening. Therefore, it is crucial to develop feasible therapeutic treatments for stroke. In this study, we tested exosomes derived from embryonic endothelial progenitor cells (eEPC) to assess their therapeutic efficacy in a rat model of ischemic stroke. Importantly, we have developed purification methods aimed at producing robust and scalable exosomes suitable for manufacturing clinical grade therapeutic exosomes. We characterized exosome cargos including RNA-seq, miRNAs targets, and proteomic mass spectrometry analysis, and we found that eEPC-exosomes were enhanced with angiogenic miRNAs (i.e., miR-126), anti-inflammatory miRNA (i.e., miR-146), and anti-apoptotic miRNAs (i.e., miR-21). The angiogenic activity of diverse eEPC-exosomes sourced from a panel of eEPC production lines was assessed in vitro by live-cell vascular tube formation and scratch wound assays, showing that several eEPC-exosomes promoted the proliferation, tube formation, and migration in endothelial cells. We further applied the exosomes systemically in a rat middle cerebral artery occlusion (MCAO) model of stroke and tested for neurological recovery (mNSS) after injury in ischemic animals. The mNSS scores revealed that recovery of sensorimotor functioning in ischemic MCAO rats increased significantly after intravenous administration of eEPC-exosomes and outpaced recovery obtained through treatment with umbilical cord stem cells. Finally, we investigated the potential mechanism of eEPC-exosomes in mitigating ischemic stroke injury and inflammation by the expression of neuronal, endothelial, and inflammatory markers. Taken together, these data support the finding that eEPCs provide a valuable source of exosomes for developing scalable therapeutic products and therapies for stroke and other ischemic diseases.

Secreted LGALS3BP facilitates distant metastasis of breast cancer

BackgroundPatients with estrogen receptor (ER)-positive breast cancer (BC) can be treated with endocrine therapy targeting ER, however, metastatic recurrence occurs in 25% of the patients who have initially been treated. Secreted proteins from tumors play important roles in cancer metastasis but previous methods for isolating secretory proteins had limitations in identifying novel targets.MethodsWe applied an in situ secretory protein labeling technique using TurboID to analyze secretome from tamoxifen-resistant (TAMR) BC. The increased expression of LGALS3BP was validated using western blotting, qPCR, ELISA, and IF. Chromatin immunoprecipitation was applied to analyze estrogen-dependent regulation of LGALS3BP transcription. The adhesive and angiogenic functions of LGALS3BP were evaluated by abrogating LGALS3BP expression using either shRNA-mediated knockdown or a neutralizing antibody. Xenograft mouse experiments were employed to assess the in vivo metastatic potential of TAMR cells and the LGALS3BP protein. Clinical evaluation of LGALS3BP risk was carried out with refractory clinical specimens from tamoxifen-treated ER-positive BC patients and publicly available databases.ResultsTAMR secretome analysis revealed that 176 proteins were secreted at least 2-fold more from MCF7/TAMR cells than from sensitive cells, and biological processes such as cell adhesion and angiogenesis were associated with the TAMR secretome. Galectin-3 binding protein (LGALS3BP) was one of the top 10 most highly secreted proteins in the TAMR secretome. The expression level of LGALS3BP was suppressed by estrogen signaling, which involves direct ERα binding to its promoter region. Secreted LGALS3BP in the TAMR secretome helped BC cells adhere to the extracellular matrix and promoted the tube formation of human umbilical vein endothelial cells. Compared with sensitive cells, xenograft animal experiments with MCF7/TAMR cells showed increased pulmonary metastasis, which completely disappeared in LGALS3BP-knockdown TAMR cells. Finally, higher levels of LGALS3BP were associated with poor prognosis in ER-positive BC patients treated with adjuvant tamoxifen in the clinic.ConclusionTAMR secretome analysis identified secretory proteins, such as LGALS3BP, that are involved in biological processes closely related to metastasis. Secreted LGALS3BP from the TAMR cells promoted adhesion of the cells to the extracellular matrix and vasculature formation, which may support metastasis of TAMR cells.

Madecassoside mitigates acute myocardial infarction injury by activating the PKCB/SPARC signaling pathway.

The current treatments and drugs of myocardial infarction (MI) remain insufficient. In recent years, natural products have garnered significant attention for their potential in treating cardiovascular diseases due to their availability and lower toxicity. Saponins, in particular, showed promising effects for cardiac protection. In this study, we investigated the therapeutic effects of the saponin compound madecassoside in the treatment of MI, and underlying molecular mechanisms. The acute MI model was established in male mice by ligation of the left anterior descending coronary artery. The mice were treated with madecassoside (20 mg· kg-1 ·d-1, i.g.) for 14 days. After sacrificing the mice, hearts were harvested for analysis. We showed that madecassoside administration significantly mitigated cardiac function decline in MI mice by promoting angiogenesis and inhibiting myocardial cell apoptosis and fibrosis. By conducting systems pharmacology and RNA sequencing, we demonstrated that madecassoside upregulated SPARC gene expression by activating protein kinase C-β (PKCB) that had a strong promoting effect on endothelial cell angiogenesis, thus playing a crucial protective role against MI. We showed that inhibition of SPARC gene significantly reduced madecassoside-stimulated migration and tube formation of endothelial cells in vitro; co-administration of the PKCB-specific inhibitor ruboxistaurin (10 mg· kg-1 ·d-1, i.g.) abolished the cardioprotective effect of madecassoside in MI mice, validating the critical role of the PKCB/SPARC signaling pathway. This study demonstrates that madecassoside regulates the PKCB/SPARC pathway, promotes the proliferation and regeneration of vascular endothelial cells, and effectively alleviates the symptoms of MI.

Inhibition of Endothelial Cell Tube Formation by Anti-Vascular Endothelial Growth Factor/Anti-Angiopoietin-2 RNA Nanoparticles.

RNA nanoparticles, derived from the packaging RNA three-way junction motif (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor, have been demonstrated to be thermodynamically and chemically stable, with promise as a nanodelivery system. Background/Objectives: A previous study showed that RNA nanoparticles with antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) inhibited cell proliferation via WST-1 assay. To further investigate the antiangiogenic potential of these RNA nanoparticles, a modified three-dimensional (3D) spheroid sprouting assay model of human umbilical vein endothelial cells was utilized in the present study. Methods: Three groups of RNA nanoparticles were evaluated, namely, pRNA-3WJ series, RNA square series (polygon-type RNA nanoparticles), and 8WJ series (multiple-way junction RNA nanoparticles), which were conjugated with a single anti-VEGF, the combination of one anti-VEGF and one anti-Ang2, or multiple anti-VEGF aptamers. The core scaffold RNA nanoparticles (without aptamers) were used as the references, and bevacizumab was used as the positive control. Results: The results demonstrated the inhibition effects of the RNA nanoparticles on endothelial cell tube formation at 67 nM in a 3D spheroid sprouting model. The results in the 3D spheroid sprouting assay are consistent with those of the WST-1 proliferation assays. Conclusions: Among the RNA nanoparticles evaluated, 3WJ-3VEGF and SQR-VEGF-Ang2 had inhibition effects equivalent to bevacizumab and were promising for anti-angiogenesis treatment.

Upregulation of OGT-mediated EZH2 O-GlcNAcylation Promotes Human Umbilical Vein Endothelial Cell Proliferation, Invasion, Migration, and Tube Formation in Gestational Diabetes Mellitus

Upregulation of OGT-mediated EZH2 O-GlcNAcylation Promotes Human Umbilical Vein Endothelial Cell Proliferation, Invasion, Migration, and Tube Formation in Gestational Diabetes Mellitus

ENO2-Regulated Glycolysis in Endothelial Cells Contributes to FGF2-Induced Retinal Neovascularization.

Ocular neovascularization is a major cause of blindness. Although fibroblast growth factor-2 (FGF2) has been implicated in the pathophysiology of angiogenesis, the underlying mechanisms remain incompletely understood. The purpose of this study was to investigate the role of FGF2 in retinal neovascularization and elucidate its underlying mechanisms. The oxygen-induced retinopathy mouse model was used to study the pathogenesis of retinal neovascularization. Immunofluorescence was used to quantify the neovascularization in retina. Data-independent acquisition proteomics were performed to quantify differentially expressed proteins in human retinal microvascular endothelial cells stimulated with FGF2 and associated pathways were analyzed. We carried out qRT-PCR and Western Blot assays to detect the expression of genes at mRNA and protein levels. The angiogenesis abilities of human retinal microvascular endothelial cells were measured by transwell, EdU and tube formation assays. FGF2 was significantly upregulated in retinal tissues of the oxygen-induced retinopathy mouse model and it markedly enhanced tube formation, migration, and proliferation abilities of human retinal microvascular endothelial cells in vitro. The proteomic analysis identified 287 differentially expressed proteins in endothelial cells in response to FGF2 stimulation, characterized by a notable upregulation of the glycolysis pathway, among which we confirmed that the enolase 2 (ENO2) levels were elevated after FGF2 stimulation, and its knockdown resulted in diminished glycolytic activity and impaired angiogenic processes. Furthermore, the use of the ENO2 inhibitor AP-Ⅲ-a4 alleviated angiogenesis in vivo and in vitro. Our findings underscore the pivotal role of ENO2-mediated glycolysis in FGF2-induced angiogenesis, suggesting that ENO2 may serve as a promising therapeutic target for managing pathological neovascularization.

Selenium-Chondroitin Sulfate Nanoparticles Inhibit Angiogenesis by Regulating the VEGFR2-Mediated PI3K/Akt Pathway.

Chondroitin sulfate (CS), a class of glycosaminoglycans covalently attached to proteins to form proteoglycans, is widely distributed in the extracellular matrix and cell surface of animal tissues. In our previous study, CS was used as a template for the synthesis of seleno-chondroitin sulfate (SeCS) through the redox reaction of ascorbic acid (Vc) and sodium selenite (Na2SeO3) and we found that SeCS could inhibit tumor cell proliferation and invasion. However, its effect on angiogenesis and its underlying mechanism are unknown. In this study, we analyzed the effect of SeCS on tube formation in vitro, based on the inhibition of tube formation and migration of human umbilical vein endothelial cells (HUVECs), and evaluated the in vivo angiogenic effect of SeCS using the chick embryo chorioallantoic membrane (CAM) assay. The results showed that SeCS significantly inhibited the angiogenesis of chicken embryo urothelium. Further mechanism analysis showed that SeCS had a strong inhibitory effect on VEGFR2 expression and its downstream PI3K/Akt signaling pathway, which contributed to its anti-angiogenic effects. In summary, SeCS showed good anti-angiogenic effects in an HUVEC cell model and a CAM model, suggesting that it may be a potential angiogenesis inhibitor.

Hypoxia-regulated miR-103-3p/FGF2 axis in adipose-derived stem cells promotes angiogenesis by vascular endothelial cells during ischemic tissue repair.

Identifying factors mediating adipose-derived stem cells (ADSCs)-induced endothelial cell angiogenesis in hypoxic skin flap tissue is critical for reconstruction. While the paracrine action of VEGF by adipose-derived stem cells (ADSCs) is established in promoting endothelial cell angiogenesis, the role of FGF2 and its regulatory mechanisms in ADSCs paracrine secretion remains unclear. We induced hypoxia and examined the expression level of FGF2 in ADSCs using ELISA, qRT-PCR, and western blotting. Proliferation of ADSCs under hypoxia was assessed using a CCK-8 assay. Co-culture experiments of hypoxia-induced ADSCs with vascular endothelial cells were conducted, and migration and tube formation abilities were evaluated through wound healing assays, transwell cell migration, and tube formation experiments. Hypoxia treatment induced significant upregulation of FGF2 expression in ADSCs, along with enhanced cell proliferation. Co-culture of hypoxia-induced ADSCs with vascular endothelial cells showed increased migration and tube formation abilities of endothelial cells. Knockdown of FGF2 inhibited these processes, while overexpression of miR-103-3p mimics in ADSCs suppressed endothelial cell migration and tube formation. FGF2 is a direct target of miR-103-3p in ADSCs. miR-103-3p/FGF2 axis regulates ADSCs on the biological activity of co-cultured vascular endothelial cells. Moreover, in the ischemic skin flap nude mouse model, ADSCs injection showed increased blood vessel formation and reduced flap necrosis, with the most significant improvement observed with ADSCs of miR-103-3p inhibitor overexpressed. Hypoxia induces paracrine secretion of FGF2 from ADSCs, which enhances endothelial cell angiogenesis. FGF2 expression is regulated by miR-103-3p in ADSCs. The miR-103-3p/FGF2 axis induces endothelial cell migration and angiogenesis and finally modulates ischemic skin flap repair in nude mice in vivo.

Functional epitope mapping of cell surface glucose-regulated protein 94: A combinatorial approach for therapeutic targeting.

Glucose-regulated protein 94 (GRP94) overexpression plays a critical role in tumor cell survival across various cancers. Previously, we developed K101.1, a fully human antibody targeting cell surface GRP94, which effectively inhibits tumor angiogenesis in colorectal cancer (CRC). This study aims to identify K101.1's interaction site and further elucidate GRP94's role in tumor angiogenesis. In an HT29 CRC xenograft mouse model, K101.1 reduced tumor growth and angiogenesis by 30% and 69%, respectively, without inducing severe toxicity. Using hydrogen‑deuterium exchange mass spectrometry, we identified the binding site of K101.1 on GRP94, analyzing 225 peptides with 94.5% sequence coverage and a redundancy score of 4.20. This revealed a linear epitope (Glu26-Ala34) as the specific binding site. The binding was further validated via enzyme-linked immunosorbent assay, and human umbilical vein endothelial cell tube formation assays using a synthetic epitope peptide corresponding to the identified epitope. Our findings suggest that this epitope is functionally involved in GRP94-mediated angiogenesis and is integral to its proangiogenic activity. By integrating epitope profiling with complementary experimental approaches, this study advances the understanding of GRP94's role in CRC angiogenesis and provides new insights for developing targeted cancer therapies and vaccines, offering promising avenues for CRC prevention and treatment.

Angiogenesis-promoting effect of SKP-SC-EVs-derived miRNA-30a-5p in peripheral nerve regeneration by targeting LIF and ANGPT2.

Ischemia and hypoxia caused by vascular injury intensify nerve damage. Skin precursor-derived Schwann cells have demonstrated an accelerated in vivo pre-vascularization of tissue-engineered nerves. Furthermore, extracellular vesicles from skin precursor-derived Schwann cells (SKP-SC-EVs) show the potential in aiding peripheral nerve regeneration. Nonetheless, the capacity of SKP-SC-EVs to facilitate nerve repair via angiogenesis remains uncertain. This study observed that SKP-SC-EVs significantly enhanced angiogenesis, evidenced by increased transparency of the tissue-engineered nerve graft and ultrasonic blood flow imaging. In vitro experiments confirmed that SKP-SC-EVs promote the proliferation, migration, and tube formation of human umbilical vein endothelial cells, a standard model for assessing angiogenic potential. Additionally, a comprehensive microRNA (miRNA) expression profile of SKP-SC-EVs was performed, leading to the identification of potential candidates through functional experiments. Among these, miR-30a-5p emerged as a significant candidate, demonstrating remarkable pro-angiogenic effects both in vivo and in vitro, akin to the effects of SKP-SC-EVs. Furthermore, luciferase reporter assay and functional experiments revealed that miR-30a-5p in SKP-SC-EVs promotes angiogenesis by targeting ANGPT2 and LIF without sufficient VEGFa. Thus, the enrichment of miR-30a-5p in SKP-SC-EVs indicates its pivotal role as a regulator of angiogenesis, presenting a promising avenue for cell-free treatment of peripheral nerve injury.

Platelet Membrane-Coated HGF-PLGA Nanoparticles Promote Therapeutic Angiogenesis and Tissue Perfusion Recovery in Ischemic Hindlimbs.

Therapeutic angiogenesis has garnered significant attention as a potential treatment strategy for lower limb ischemic diseases. Although hepatocyte growth factor (HGF) has been identified as a key promoter of therapeutic angiogenesis, its clinical application is limited due to its short half-life. In this study, we successfully developed and characterized platelet membrane-coated HGF-poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). These nanoparticles demonstrated enhanced capabilities to promote endothelial cell (EC) proliferation, migration, and tube formation in vitro. Additionally, their efficacy in improving tissue perfusion and promoting angiogenesis was confirmed in a hindlimb ischemia rat model. Our findings suggest that platelet membrane-coated HGF-PLGA-NPs could serve as a promising therapeutic approach for enhancing angiogenesis and restoring tissue perfusion in ischemic conditions.