Heme: A link between hemorrhage and retinopathy of prematurity progression.

Neovascularization is implicated in the pathology of retinopathy of prematurity (ROP), diabetic retinopathy (DR), and age-related macular degeneration (AMD), which are the leading causes of blindness worldwide. In our work, we analyzed how heme released during hemorrhage affects hypoxic response and neovascularization. Our retrospective clinical analysis demonstrated, that hemorrhage was associated with more severe retinal neovascularization in ROP patients. Our heme-stimulated human retinal pigment epithelial (ARPE-19) cell studies demonstrated increased expression of positive regulators of angiogenesis, including vascular endothelial growth factor-A (VEGFA), a key player of ROP, DR and AMD, and highlighted the activation of the PI3K/AKT/mTOR/VEGFA pathway involved in angiogenesis in response to heme. Furthermore, heme decreased oxidative phosphorylation in the mitochondria, augmented glycolysis, facilitated HIF-1α nuclear translocation, and increased VEGFA/GLUT1/PDK1 expression suggesting HIF-1α-driven hypoxic response in ARPE-19 cells without effecting the metabolism of reactive oxygen species. Inhibitors of HIF-1α, PI3K and suppression of mTOR pathway by clinically promising drug, rapamycin, mitigated heme-provoked cellular response. Our data proved that oxidatively modified forms of hemoglobin can be sources of heme to induce VEGFA during retinal hemorrhage. We propose that hemorrhage is involved in the pathology of ROP, DR, and AMD.

VEPTP inhibition with an extracellular domain targeting antibody did not restore albuminuria in a mouse model of diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. DKD is a heterogeneous disease with complex pathophysiology where early endothelial dysfunction is associated with disease progression. The Tie2 receptor and Angiopoietin 1 and 2 ligands are critical for maintaining endothelial cell permeability and integrity. Tie2 signaling is negatively regulated by the endothelial specific transmembrane receptor Vascular Endothelial Protein Tyrosine Phosphatase (VEPTP). Genetic deletion of VEPTP protects from hypertension and diabetes induced renal injury in a mouse model of DKD. Here, we show that VEPTP inhibition with an extracellular domain targeting VEPTP antibody induced Tie2 phosphorylation and improved VEGF-A induced vascular permeability both in vitro and in vivo. Treatment with the VEPTP blocking antibody decreased the renal expression of endothelial activation markers (Angpt2, Edn1, and Icam1) but failed to improve kidney function in db/db uninephrectomized ReninAAV DKD mice.

Dipeptidyl peptidase-4 marks distinct subtypes of human adipose stromal/stem cells with different hepatocyte differentiation and immunoregulatory properties.

BACKGROUND: Human adipose-derived stromal/stem cells (hASCs) play important roles in regenerative medicine and numerous inflammatory diseases. However, their cellular heterogeneity limits the effectiveness of treatment. Understanding the distinct subtypes of hASCs and their phenotypic implications will enable the selection of appropriate subpopulations for targeted approaches in regenerative medicine or inflammatory diseases. METHODS: hASC subtypes expressing dipeptidyl peptidase-4 (DPP4) were identified via fluorescence-activated cell sorting (FACS) analysis. DPP4 expression was knocked down in DPP4+ hASCs via DPP4 siRNA. The capacity for proliferation, hepatocyte differentiation, inflammatory factor secretion and T-cell functionality regulation of hASCs from DPP4-, DPP4+, and control siRNA-treated DPP4+ hASCs and DPP4 siRNA-treated DPP4+ hASCs were assessed. RESULTS: DPP4+ hASCs and control siRNA-treated DPP4+ hASCs presented a lower proliferative capacity but greater hepatocyte differentiation capacity than DPP4- hASCs and DPP4 siRNA-treated DPP4+ hASCs. Both DPP4+ hASCs and DPP4- hASCs secreted high levels of vascular endothelial growth factor-A (VEGF-A), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6), whereas the levels of other factors, including matrix metalloproteinase (MMP)-1, eotaxin-3, fractalkine (FKN, CX3CL1), growth-related oncogene-alpha (GRO-alpha, CXCL1), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP)-1beta, and macrophage colony-stimulating factor (M-CSF), were significantly greater in the supernatants of DPP4+ hASCs than in those of DPP4- hASCs. Exposure to hASC subtypes and their conditioned media triggered changes in the secreted cytokine profiles of T cells from healthy donors. The percentage of functional T cells that secreted factors such as MIP-1beta and IL-8 increased when these cells were cocultured with DPP4+ hASCs. The percentage of polyfunctional CD8+ T cells that secreted multiple factors, such as IL-17A, tumour necrosis factor alpha (TNF-α) and TNF-ß, decreased when these cells were cocultured with supernatants derived from DPP4+ hASCs. CONCLUSIONS: DPP4 may regulate proliferation, hepatocyte differentiation, inflammatory cytokine secretion and T-cell functionality of hASCs. These data provide a key foundation for understanding the important role of hASC subpopulations in the regulation of T cells, which may be helpful for future immune activation studies and allow them to be customized for clinical application.

Gene Therapeutic Drug pCMV-VEGF165 Plasmid ('Neovasculgen') Promotes Gingiva Soft Tissue Augmentation in Rabbits.

Currently, an increasing number of patients are undergoing extensive surgeries to restore the mucosa of the gums in the area of recessions. The use of a connective tissue graft from the palate is the gold standard of such surgical treatment, but complications, especially in cases of extensive defects, have led to the development of approaches using xenogeneic collagen matrices and methods to stimulate their regenerative and vasculogenic potential. This study investigated the potential of a xenogeneic scaffold derived from porcine skin Mucoderm and injections of the pCMV-VEGF165 plasmid ('Neovasculgen') to enhance soft gingival tissue volume and vascularization in an experimental model in rabbits. In vitro studies demonstrated the biocompatibility of the matrix and plasmid with gingival mesenchymal stem cells, showing no toxic effects and supporting cell viability and metabolic activity. In the in vivo experiment, the combination of Mucoderm and the pCMV-VEGF165 plasmid (0.12 mg) synergistically promoted tissue proliferation and vascularization. The thickness of soft tissues at the implantation site significantly increased with the combined application (3257.8 ± 1093.5 µm). Meanwhile, in the control group, the thickness of the submucosa was 341.8 ± 65.6 µm, and after the implantation of only Mucoderm, the thickness of the submucosa was 2041.6 ± 496.8 µm. Furthermore, when using a combination of Mucoderm and the pCMV-VEGF165 plasmid, the density and diameter of blood vessels were notably augmented, with a mean value of 226.7 ± 45.9 per 1 mm2 of tissue, while in the control group, it was only 68.3 ± 17.2 per 1 mm2 of tissue. With the application of only Mucoderm, it was 131.7 ± 37.1 per 1 mm2 of tissue, and with only the pCMV-VEGF165 plasmid, it was 145 ± 37.82 per 1 mm2 of the sample. Thus, the use of the pCMV-VEGF165 plasmid ('Neovasculgen') in combination with the xenogeneic collagen matrix Mucoderm potentiated the pro-proliferative effect of the membrane and the pro-vascularization effect of the plasmid. These results indicate the promising potential of this innovative approach for clinical applications in regenerative medicine and dentistry.

Poor Response to Bevacizumab Correlates With Higher IL-6 and IL-8 Aqueous Cytokines in AMD.

Purpose: To evaluate the effect of intravitreal bevacizumab on aqueous levels of a panel of 12 inflammatory cytokines in patients with neovascular age-related macular degeneration (nAMD) and correlate response to treatment, as measured by change in the central subfovea thickness (CST), with cytokine levels. Methods: Thirty-three treatment-naïve patients with nAMD received a loading dose of intravitreal bevacizumab consisting of three injections at six weekly intervals. The aqueous samples prior to the first (baseline), second (week 6), and third (week 12) injections were analyzed for cytokine levels. Participants were subgrouped based on changes in CST on spectral-domain optical coherence tomography (SD-OCT) at 12 weeks. Group 1 included patients with a decrease in CST (responders; n = 27). Group 2 included patients who had no decrease in CST (poor responders; n = 6). Results: Aqueous IL-8 was the only cytokine to demonstrate a significant difference in levels between responders and poor responders, with higher interleukin-8 (IL-8) at week 12 in the poor responder group. Aqueous IL-6 and IL-8 levels showed a positive correlation with CST on SD-OCT (Spearman r = 0.45 and 0.55, respectively). There was a temporal increase overall in cytokine concentration accompanying bevacizumab treatment. Conclusions: Aqueous IL-6 and IL-8 may be important markers of treatment response or poor response in nAMD. Future therapeutic strategies may include targeted treatment against both vascular endothelial cell growth factor (VEGF) and IL-6 and/or IL-8 in patients who do not respond to anti-VEGF treatment alone.

Chinese Herbal Compound Xiaoliu Pingyi Recipe Inhibits the Growth of Lung Adenocarcinoma by Regulating the Tumor Vascular Microenvironment.

BACKGROUND: The traditional Chinese medicine (TCM) Xiaoliu Pingyi recipe (XLPYR) has been clinically used for several decades, demonstrating favorable therapeutic effects. However, the underlying regulatory mechanisms remain unclear. The aim of this study was to explore the anti-tumor effects of XLPYR and its regulatory role in the vascular microenvironment through in vivo and in vitro experiment. MATERIALS AND METHODS: In the in vivo study, a C57BL/6J mouse model of lung adenocarcinoma (LUAD) allografts was established, and various interventions were administered for 14 days (Model group: administered normal saline via oral gavage; Pemetrexed (PEM) group: intraperitoneally injected with a solution of pemetrexed, once every 3d; XLPYR group: administered XLPYR via oral gavage; Combination (COMBI) group: received XLPYR via oral gavage simultaneously with intraperitoneal injection of pemetrexed solution). Tumor volume and weight were then compared among the groups. The impact of XLPYR on the tumor vascular microenvironment was assessed using immunohistochemistry staining. In the in vitro study, XLPYR-containing serum was prepared by oral administration to SD rats. The CCK-8 assay evaluated the effect of the serum on the proliferation of normal lung epithelial BEAS-2B cells and LUAD A549 cells, determining the optimal intervention concentrations. The cell migration and invasion abilities were evaluated using the wound-healing assay and Transwell assay, respectively. Finally, ELISA assay measured VEGF secretion levels in the LUAD cell supernatant, and RT-qPCR and Western Blot were employed to detect differences in HIF-1α, VEGFA, Ang-2, and PI3K/Akt mRNA and protein expression levels in both in vivo and in vitro experiments. RESULTS: In the in vivo study, XLPYR significantly inhibited the growth of mice LUAD allografts, with enhanced anti-tumor effects observed with prolonged drug intervention. Immunohistochemistry staining revealed reduced MVD and increased pericyte coverage in all intervention groups. Regarding vascular function, FITC-Dextran extravasation in the tumor tissues of the Model group was significantly higher than in the intervention groups, particularly with lower extravasation in the COMBI group compared to the PEM group. In the in vitro study, XLPYR demonstrated a time- and concentration-dependent inhibitory effect on LUAD cells, and with greater sensitivity in inhibiting LUAD cells compared to BEAS-2B cells. The wound-healing assay and Transwell assay confirmed that XLPYR significantly suppressed the migration and invasion abilities of LUAD cells. ELISA experiments further revealed a significant decrease in VEGF expression in the supernatant of each intervention group. RT-qPCR and Western Blot results showed consistent findings between the in vivo and in vitro experiments. HIF-1α, VEGFA, and Ang-2 mRNA and protein expression levels were significantly downregulated in the PEM group, XLPYR group, and COMBI group. There were no significant differences in the expression of PI3K and Akt mRNA and total protein, but the expression levels of phosphorylated p-PI3K and p-Akt were notably downregulated. CONCLUSION: XLPYR significantly inhibited C57BL/6J mouse LUAD allograft growth and improved the vascular microenvironment, thereby intervening in tumor angiogenesis and inducing vascular normalization. It suppressed LUAD cell proliferation, migration, and invasion, while reducing VEGF concentration in the cell supernatant. The regulatory mechanism may involve inhibiting PI3K/Akt protein phosphorylation and downregulating angiogenesis-related factors, such as HIF-1α, VEGF, and Ang-2.

Bone marrow stromal and anterior cruciate ligament remnant cell co-culture-derived extracellular vesicles promote cell activity in both cell types.

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co-culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell-cell interactions, act as the active components, improving graft maturation in this co-culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC-only culture), co-culture (ACL remnant cells and BMSCs, CM) and co-culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC-EV) and co-culture (CM-EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF-ß)-, vascular endothelial growth factor (VEGF)-, collagen synthesis- and tenogenesis-related genes. However, CM-induced effects were reversed by the CM ∆ EVs treatment. CM-EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC-EV and non-EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis-, TGF-ß-, VEGF- and tenogenesis-related genes in both cell types.

[Tujia medicine Toddalia asiatica improves synovial pannus in rats with collagen-induced arthritis through the PI3K/Akt signaling pathway].

OBJECTIVE: To investigate the therapeutic mechanism of Tujia medicine Toddalia asiatica alcohol extract (TAAE) for synovial pannus formation in rats with college-induced arthritis (CIA). METHODS: Sixty male SD rats were randomized into normal control group, CIA model group, TGT group, 3 TAAE treatment groups at low, medium and high doses (n=10). Except for those in the normal control group, all the rats were subjected to CIA modeling using a secondary immunization method and treatment with saline, TGT or TAAE by gavage once daily for 35 days. The severity of arthritis was assessed using arthritis index (AI) score, and knee joint synovium pathologies were examined with HE staining. Serum levels of TNF-α, IL-6, and IL-1ß were detected with ELISA; the protein expressions of PI3K, Akt, p-PI3K, p-Akt, VEGF, endostatin, HIF-1α, MMP1, MMP3, and MMP9 in knee joint synovial tissues were determined using Western blotting, and the mRNA expressions of TNF­α, IL-6, IL-1ß, VEGF, HIF-1α, PI3K, and Akt were detected with RT-PCR. RESULTS: Treatment of CIA rat models with TAAE and TGT significantly alleviated paw swelling, lowered AI scores, and reduced knee joint pathology, neoangiogenesis, and serum levels of inflammatory factors. TAAE treatment obviously increased endostatin protein expression, downregulated p-PI3K, p-Akt, MMP1, MMP3, MMP9, VEGF, and HIF-1α proteins, and reduced TNF­α, IL-6, IL-1ß, PI3K, Akt, VEGF, and HIF-1α mRNA levels in the synovial tissues, and these changes were comparable between high-dose TAAE group and TGT group. CONCLUSION: TAAE can improve joint symptoms and inhibit synovial pannus formation in CIA rats by regulating the expressions of HIF-1α, VEGF, endostatin, MMP1, MMP3, and MMP9 via the PI3K/Akt signalling pathway.

[Pharmacokinetics of intravitreal anti-VEGF drugs in vitrectomized eyes].

Neovascular retinal diseases pose a significant burden, often resulting in visual impairment. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs serves as the primary therapeutic approach. Nonetheless, certain patients necessitate continued anti-VEGF treatment post-vitrectomy or other ocular surgeries. Emerging evidence suggests that variations in surgical techniques and postoperative vitreous cavity management may induce distinct intraocular pharmacokinetics (PK) of anti-VEGF agents following vitrectomy, prompting potential adjustments in therapeutic strategies. This review offers a thorough examination of the pharmacokinetic determinants impacting anti-VEGF drugs and their intraocular dynamics post-vitrectomy.

Anti-Inflammatory and Anti-(Lymph)angiogenic Properties of an ABCB5+ Limbal Mesenchymal Stem Cell Population.

Corneal transparency and avascularity are essential for vision. The avascular cornea transitions into the vascularized conjunctiva at the limbus. Here, we explore a limbal stromal cell sub-population that expresses ABCB5 and has mesenchymal stem cell characteristics. Human primary corneal stromal cells were enriched for ABCB5 by using FACS sorting. ABCB5+ cells expressed the MSC markers CD90, CD73, and CD105. ABCB5+ but not ABCB5- cells from the same donor displayed evidence of pluripotency with a significantly higher colony-forming efficiency and the ability of trilineage differentiation (osteogenic, adipogenic, and chondrogenic). The ABCB5+ cell secretome demonstrated lower levels of the pro-inflammatory protein MIF (macrophage migration inhibitory factor) as well as of the pro-(lymph)angiogenic growth factors VEGFA and VEGFC, which correlated with reduced proliferation of Jurkat cells co-cultured with ABCB5+ cells and decreased proliferation of blood and lymphatic endothelial cells cultured in ABCB5+ cell-conditioned media. These data support the hypothesis that ABCB5+ limbal stromal cells are a putative MSC population with potential anti-inflammatory and anti-(lymph)angiogenic effects. The therapeutic modulation of ABCB5+ limbal stromal cells may prevent cornea neovascularization and inflammation and, if transplanted to other sites in the body, provide similar protective properties to other tissues.

Role of CD44-Positive Extracellular Vesicles Derived from Highly Metastatic Mouse Mammary Carcinoma Cells in Pre-Metastatic Niche Formation.

Malignant breast cancers pose a notable challenge when it comes to treatment options. Recently, research has implicated extracellular vesicles (EVs) secreted by cancer cells in the formation of a pre-metastatic niche. Small clumps of CD44-positive breast cancer cells are efficiently transferred through CD44-CD44 protein homophilic interaction. This study aims to examine the function of CD44-positive EVs in pre-metastatic niche formation in vitro and to suggest a more efficacious EV formulation. We used mouse mammary carcinoma cells, BJMC3879 Luc2 (Luc2 cells) as the source of CD44-positive EVs and mouse endothelial cells (UV2 cells) as the recipient cells in the niche. Luc2 cells exhibited an enhanced secretion of EVs expressing CD44 and endothelial growth factors (VEGF-A, -C) under 20% O2 (representative of the early stage of tumorigenesis) compared to its expression under 1% O2 (in solid tumor), indicating that pre-metastatic niche formation occurs in the early stage. Furthermore, UV2 endothelial cells expressing CD44 demonstrated a high level of engulfment of EVs that had been supplemented with hyaluronan, and the proliferation of UV2 cells occurred following the engulfment of EVs. These results suggest that anti-VEGF-A and -C encapsulated, CD44-expressing, and hyaluronan-coated EVs are more effective for tumor metastasis.

Pravastatin Protects Cytotrophoblasts from Hyperglycemia-Induced Preeclampsia Phenotype.

There are no effective therapies to prevent preeclampsia (PE). Pravastatin shows promise by attenuating processes associated with PE such as decreased cytotrophoblast (CTB) migration, aberrant angiogenesis, and increased oxidative stress. This study assesses the effects of pravastatin on hyperglycemia-induced CTB dysfunction. METHODS: Human CTB cells were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with pravastatin (1 µg/mL), while others were cotreated with pravastatin and glucose. The expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) mRNA, vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured. CTB migration was assayed using a CytoSelect migration assay kit. Statistical comparisons were performed using an analysis of variance with Duncan's post hoc test. RESULTS: The hyperglycemia-induced downregulation of uPA was attenuated in CTB cells pretreated with pravastatin at glucose levels > 200 mg/dL and cotreated at glucose levels > 300 mg/dL (p < 0.05). Hyperglycemia-induced decreases in VEGF and PlGF and increases in sEng and sFlt-1 were attenuated in both the pretreatment and cotreatment samples regardless of glucose dose (p < 0.05). Pravastatin attenuated hyperglycemia-induced dysfunction of CTB migration. CONCLUSIONS: Pravastatin mitigates stress signaling responses in hyperglycemic conditions, weakening processes leading to abnormal CTB migration and invasion associated with PE in pregnancy.

Comparative Investigation of Photobiomodulation in Diabetes-Impaired Alveolar Bone Healing: A Histomorphometrical and Molecular Study.

Objective: Diabetes mellitus is increasing worldwide. Photobiomodulation (PBM) is proposed as a therapeutic method in various medical concerns. This study aimed to compare the effects of PBM at the wavelengths of 660, 808, or 660 + 808 nm on alveolar bone healing in diabetic rats. Methods: Bilateral maxillary first molars were extracted from diabetic Wistar rats (n = 36). Right-sided sockets were treated by an In-Ga-Al-P laser at 660 nm (7.2 J/cm2, 24 s; DM660), Ga-Al-As laser at 808 nm (7 J/cm2, 14 s; DM808), or a combination of these two sets (DM-dual) (n = 12). Left sides served as controls. On days 7 or 14, specimens were assigned for histomorphometric or real-time PCR analysis of runt-related transcription factor 2, osteocalcin, collagen I, and vascular endothelial growth factor expression. Results: Irradiated sockets of groups DM-808 and DM-dual showed a significant increase in bone tissue and blood vessel establishment as compared to DM-660. Further, group DM-dual exhibited the least amount of fibrotic tissue as compared to the other groups. Conclusions: Within our study limits, the present experiment suggested PBM at 808 nm, alone or combined with 660 nm irradiation, could promote alveolar bone healing, along with minimal fibrosis induction, in diabetic rats.

Proteome of plasma extracellular vesicles as a source of colorectal cancer biomarkers.

The search for minimally invasive methods for diagnostics of colorectal cancer (CRC) is the most important task for early diagnostics of the disease and subsequent successful treatment. Human plasma represents the main type of biological material used in the clinical practice; however, the complex dynamic range of substances circulating in it complicates determination of CRC protein markers by the mass spectrometric (MS) method. Studying the proteome of extracellular vesicles (EVs) isolated from human plasma represents an attractive approach for the discovery of tissue-secreted CRC markers. We performed shotgun mass spectrometry analysis of EV samples obtained from plasma of CRC patients and healthy volunteers. This MS analysis resulted in identification of 370 proteins (which were registered by at least two peptides). Stable isotope-free relative quantitation identified 55 proteins with altered abundance in EV samples obtained from plasma samples of CRC patients as compared to healthy controls. Among the EV proteins isolated from blood plasma we found components involved in cell adhesion and the VEGFA-VEGFR2 signaling pathway (TLN1, HSPA8, VCL, MYH9, and others), as well as proteins expressed predominantly by gastrointestinal tissues (polymeric immunoglobulin receptor, PIGR). The data obtained using the shotgun proteomic profiling may be added to the panel for targeted MS analysis of EV-associated protein markers, previously developed using CRC cell models.

Specific mode electroacupuncture stimulation opens the blood-brain barrier of the infarcted border zone in rats during MCAO/R recovery via modulation of tight junction protein expression by VEGFA and NF-κB.

The blood-brain barrier (BBB) strictly limits the entry of most exogenous therapeutic drugs into the brain, which brings great challenges to the drug treatment of refractory central diseases, including the treatment of ischemic stroke. Our previous studies have shown that specific mode electroacupuncture stimulation (SMES) can temporarily open the BBB, but with the mechanisms largely unknown. This study explored whether SMES opens the BBB in the infarcted border zone of rats during middle cerebral artery occlusion/reperfusion recovery, and whether this is related to p65 or vascular endothelial growth factor A (VEGFA) modulation of tight junction protein expression through in vivo and in vitro studies. Evans blue, FITC-dextran, mouse-derived nerve growth factor (NGF), and transendothelial electrical resistance values were used to evaluate the permeability of the BBB. Additionally, microvascular endothelial cells and astrocytes were utilized for in vitro study. Immunofluorescence, immunohistochemistry, western blot, and ELISA were employed to assess related protein expression. SMES significantly increased vascular permeability for Evans blue and NGF in the infarcted border zone, and increased the expression of VEGFA by activating p-p65, thereby reducing the expression of tight junction proteins Occludin and ZO-1. Correspondingly, oxygen glucose deprivation/reoxygenation activated p-p65 in and induced VEGFA secretion from astrocytes in vitro. Their conditioned medium reduced the expression of Occludin in bEnd.3 cells and increased the permeability of FITC-dextran. The mechanism of SMES opening infarcted border zone BBB is partly related to its actions on p65, VEGFA, and tight junction proteins.

Exploring angiogenic pathways in breast cancer: Clinicopathologic correlations and prognostic implications based on gene expression profiles from a large-scale genomic dataset.

BACKGROUND: Angiogenesis inhibitors targeting VEGF, or its receptors have consistently produced disappointing clinical outcomes in breast cancer. Therefore, there is an urgent need to explore alternative angiogenic pathways in breast cancer. This study aimed to describe the gene expression of pivotal pro-angiogenic genes in breast cancer and to further analyze the associations with the clinicopathologic tumor features, prognostic factors, and overall survival. Such findings would expand the understanding of the role of different angiogenic pathways in breast cancer pathogenesis and identify patients at risk of more aggressive disease who could be eligible for intense treatment regimens. Additionally, exploring angiogenic pathways helps identify new potential drug targets for breast cancer. METHODS: The mRNA expression levels for eight pro-angiogenic genes [VEGFA, HGF, FGF1, FGF2, ANGPT1, ANGPT2, PDGFA, and PDGFB] were obtained from the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) dataset available at cBioPortal public domain. Pertinent demographic and tumor information were retrieved. RESULTS: VEGFA and ANGPT2 genes had the highest expression levels with average mRNA log intensities of 7.18±0.7 and 7.11±0.53, respectively. VEGFA expression was not correlated with the expression of other pro-angiogenic genes, the clinicopathologic tumor features, and the overall survival of patients. FGF1, ANGPT1, and PDGFA mRNA levels were negatively correlated with the age of patients at diagnosis. The expression of FGF1 and FGF2 correlated inversely with tumor size and the Nottingham Prognostic Index (p = 0.03 and p = 0.002, respectively). Expression of HGF was significantly associated with advanced tumor stage (p<0.05). Expression of ANGPT1 and ANGPT2 was associated with hormone receptor-negative status and the non-luminal subtypes. PDGFB expression was significantly higher in patients with high-grade disease and HER2-positive status. Patients with high expression status of ANGPT2 and PDGFB had significantly reduced overall survival compared to those with low expression levels of these genes (p = 0.004 and p = 0.0001, respectively). CONCLUSIONS: In this dataset of patients with breast cancer, the expression levels of 8 different pro-angiogenic genes revealed remarkable differences in terms of their association with clinicopathologic tumor characteristics and prognosis. The expression of ANGPTs and PDGFs was associated with adverse tumor features, worse prognosis, and reduced survival in patients. Targeting ANGPTs and PDGF pathways could provide new insights for effective anti-angiogenic drugs in breast cancer.

MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathway.

OBJECTIVE: To explore whether miR-199a-5p regulated BBB integrity through PI3K/Akt pathway after ischemia stroke. METHODS: Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion(MCAO) were used in experiment. The Ludmila Belayev 12-point scoring was used to measure the neurological function of MCAO rats. The Evans Blue Stain, immunofluorescence staining, western-blotting and RT-PCR were performed to evaluate the effects of miR-199a-5p mimic on BBB integrity in rats following MCAO. RESULTS: The result suggested that miR-199a-5p mimic treatment possessed the potential to boost proprioception and motor activity of MCAO rats. MiR-199a-5p decreased the expression of PIK3R2 after MCAO, activated Akt signaling pathway, and increased the expression of Claudin-5 and VEGF in the ischemic penumbra. Furthermore, miR-199a-5p alleviated inflammation after cerebral ischemia. BBB leakage and neurocyte apoptosis were cut down in MCAO rats treated with miR-199a-5p mimic. CONCLUSIONS: MiR-199a-5p mimic decreased the expression of PIK3R2 and activated Akt signaling pathway after ischemia stroke, reduced the expression of inflammatory cytokines, and attenuated BBB disruption after ischemic stroke.

New Insight in Using of Mesenchyme Stem Cell Conditioning Medium for the Impaired Muscle related Biomarkers: In vivo Study with Rat Model.

AIMS AND OBJECTIVES: This study aimed to investigate the effects of Umbilical Cord Mesencymal Stem Cell Conditioning Medium (UC MSC-CM) administration on body weight recovery and the level of four molecular biomarkers, namely Superoxide Dismutase (SOD), vascular Endothelial Growth Factor (VEGF), C-Reactive Protein (CRP), and myostatin. MATERIALS AND METHODS: Secretome was injected intramuscularly twice at 1.5 mL (day 7 and 14) into the right thigh of high-dose, short-term galactose-induced aging rats. The data of day 7 (before) and day 21 (after the administration) were evaluated. The body weights and the four biomarkers were measured before (day 7) and after intervention (day 21). RESULTS: This study showed that the UC MSC-CM intramuscular administrations did not influence body weight regeneration. However, it could increase SOD and VEGF levels and decrease CRP and myostatin levels. CONCLUSION: Treatment with UC MSC-CM is a promising and potential agent in treating sarcopenia.

Résumé Buts et objectifs:Cette étude visait à examiner les effets de l'administration d'un milieu de conditionnement de cellules souches mésencéphaliques de cordon ombilical (UC MSC-CM) sur la récupération du poids corporel et le niveau de quatre biomarqueurs moléculaires, à savoir la superoxyde dismutase (SOD), le facteur de croissance endothéliale vasculaire (VEGF), la protéine C-réactive (CRP) et la myostatine.Matériels et méthodes:Le sécrétome (UC MSC-CM) a été injecté par voie intramusculaire deux fois à 1,5 ml (jour 7 et 14) dans la cuisse droite de rats vieillissant à forte dose et à court terme induits par le galactose. Les données du jour 7 (avant) et du jour 21 (après l'administration) ont été évaluées. Le poids corporel et les quatre biomarqueurs ont été mesurés avant (jour 7) et après l'intervention (jour 21).Résultats:Cette étude a montré que les administrations intramusculaires de CSM-CM d'UC n'ont pas influencé la régénération du poids corporel. Cependant, elle a pu augmenter les niveaux de SOD et de VEGF et diminuer les niveaux de CRP et de myostatine.Conclusion:Le traitement par UC MSC-CM est un agent prometteur et potentiel dans le traitement de la sarcopénie.

Enhanced efficacy of combined VEGFR peptide-drug conjugate and anti-PD-1 antibody in treating hepatocellular carcinoma.

This study aimed to design a VEGFR-targeting peptide-drug conjugate with the ability to decrease tumor burden and suppress tumor angiogenesis, and to further evaluate the therapeutic effect of anti-PD-1 antibody in HCC therapy. A VEGFR-targeting peptide VEGF125 - 136 (QR) was conjugated with a lytic peptide (KLU) to form a peptide-drug conjugate QR-KLU. And the efficacy of QR-KLU in combination with anti-PD-1 antibody for HCC therapy in vivo and in vitro were evaluated. QR-KLU inhibited the proliferation and migration of mouse HCC cell line (Hepa1-6) cells under normoxic and hypoxic conditions in a dose-dependent manner. In the subcutaneous Hepa1-6 tumor model, QR-KLU combined with the anti-PD-1 antibody substantially inhibited tumor growth, promoted tumor necrosis, and prolonged the survival time of tumor-bearing mice. QR-KLU substantially inhibited hypoxia-induced expression of VEGF, promoted tumor vascular normalization, and increased cluster of differentiation 8+ (CD8+) T cell infiltration in the tumor. In addition, QR-KLU and anti-PD-1 antibody demonstrated a strong synergistic effect in promoting the activation of intratumoral CD8+ T cells, reducing the expression of immune-inhibitory factors, and increasing the expression of immune-stimulatory factors. This study proposed a novel approach for enhancing the efficacy of anti-PD-1 antibody using a VEGFR-targeting peptide-drug conjugate in HCC therapy.

Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood-brain barrier integrity.

The neurovascular unit (NVU) is a complex multicellular structure that helps maintain cerebral homeostasis and blood-brain barrier (BBB) integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration, the underlying molecular mechanisms remain unclear. Here, we use zebrafish embryos carrying a mutation in Scavenger Receptor B2, a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells (RGCs), to investigate the interplay among different NVU components. Through live imaging and genetic manipulations, we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling, thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling, leading to severe vascular defects, hemorrhages, and a leaky BBB. Altogether, our findings provide insights into NVU formation and function and offer avenues for investigating diseases involving white matter defects and vascular abnormalities.