OBJECTIVE: To explore the mechanism of antibiotic delivery system targeting bacterial biofilm with linezolid (LZD) based on ε-poly- L-lysine (ε-PLL) and cyclodextrin (CD) (ε-PLL-CD-LZD), aiming to enhance antibiotic bioavailability, effectively penetrate and disrupt biofilm structures, and thereby improve the treatment of bone and joint infections. METHODS: ε-PLL-CD-LZD was synthesized via chemical methods. The grafting rate of CD was characterized using nuclear magnetic resonance. In vitro biocompatibility was evaluated through live/dead cell staining after co-culturing with mouse embryonic osteoblast precursor cells (MC3T3-E1), human umbilical vein endothelial cells, and mouse embryonic fibroblast cells (3T3-L1). The biofilm-enrichment capacity of ε-PLL-CD-LZD was assessed using Staphylococcus aureus biofilms through enrichment studies. Its biofilm eradication efficacy was investigated via minimum inhibitory concentration (MIC) determination, scanning electron microscopy, and live/dead bacterial staining. A bone and joint infection model in male Sprague-Dawley rats was established to validate the antibacterial effects of ε-PLL-CD-LZD. RESULTS: In ε-PLL-CD-LZD, the average grafting rate of CD reached 9.88%. The cell viability exceeded 90% after co-culturing with three types cells. The strong biofilm enrichment capability was observed with a MIC of 2 mg/L. Scanning electron microscopy observations revealed the effective disruption of biofilm structure, indicating potent biofilm eradication capacity. In vivo rat experiments demonstrated that ε-PLL-CD-LZD significantly reduced bacterial load and infection positivity rate at the lesion site ( P<0.05). CONCLUSION The ε-PLL-CD antibiotic delivery system provides a treatment strategy for bone and joint infections with high clinical translational significance. By effectively enhancing antibiotic bioavailability, penetrating, and disrupting biofilms, it demonstrated significant anti-infection effects in animal models.
Biofilms/drug effects* ; Animals ; Anti-Bacterial Agents/pharmacology* ; Polylysine/chemistry* ; Cyclodextrins/administration & dosage* ; Humans ; Linezolid/pharmacology* ; Staphylococcus aureus/physiology* ; Rats, Sprague-Dawley ; Mice ; Rats ; Male ; Drug Delivery Systems ; Staphylococcal Infections/drug therapy* ; Microbial Sensitivity Tests ; Human Umbilical Vein Endothelial Cells ; Osteoblasts/cytology*

Meniscus injuries are widespread and the available treatments do not offer enough healing potential. Here, we provide critical support for using pigs as a biological model for meniscal degeneration and the development of cutting-edge therapies in orthopedics. We present a single-cell transcriptome atlas of the meniscus, consisting of cell clusters corresponding to four major cell types: chondrocytes, endothelial cells, smooth muscle cells, and immune cells. Five distinct chondrocyte subclusters (CH0‒CH4) were annotated, of which only one was widespread in both the red and white zones, indicating a major difference in the cellular makeup of the zones. Subclusters distinct to the white zone appear responsible for cartilage-specific matrix deposition and protection against adverse microenvironmental factors, while those in the red zone exhibit characteristics of mesenchymal stem cells and are more likely to proliferate and migrate. Additionally, they induce remodeling actions in other chondrocyte subclusters and promote the proliferation and maturation of endothelial cells, inducing healing and vascularization processes. Considering that they have substantial remodeling capabilities, these subclusters should be of great interest for tissue engineering studies. We also show that the cellular makeup of the pig meniscus is comparable to that of humans, which supports the use of pigs as a model in orthopedic therapy development.
Animals ; Swine ; Chondrocytes/physiology* ; Single-Cell Analysis ; Meniscus/blood supply* ; Endothelial Cells/cytology* ; Transcriptome ; Mesenchymal Stem Cells/cytology* ; Neovascularization, Physiologic ; Cell Proliferation

Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/ leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.
Cell Proliferation ; Cells, Cultured ; Cytokines/biosynthesis* ; Female ; Human Umbilical Vein Endothelial Cells/radiation effects* ; Humans ; Inflammation/etiology* ; Leptin/pharmacology* ; Mesenchymal Stem Cells/physiology* ; Neovascularization, Physiologic/physiology* ; Placenta/cytology* ; Pregnancy ; STAT3 Transcription Factor/genetics* ; Transcription Factor RelA/genetics* ; X-Rays

Objective: To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells (CCECs) from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction (ED). METHODS: The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with immunomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor (VWF) in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, the purity of the CCECs determined by flow cytometry, and the proliferation of the cells measured with CCK-8 and growth curves. RESULTS: After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like flagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1－2 days, in the logarithmic growth phase with a rapid rate at 3－4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beads combined with cloning cylinders was up to (91.9±3.75)%. CONCLUSIONS High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.
Animals ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; chemistry ; cytology ; physiology ; Erectile Dysfunction ; pathology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; Male ; Penis ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; Rats ; Rats, Sprague-Dawley ; Sincalide ; analysis ; von Willebrand Factor ; analysis

OBJECTIVEThis study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish.

METHODSThe in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels.

RESULTSR1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs.

CONCLUSIONR1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

Animals ; Blood Vessels ; pathology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Collagen ; pharmacology ; Disease Models, Animal ; Drug Combinations ; Ginsenosides ; chemistry ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; physiology ; Humans ; Laminin ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Proteoglycans ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Zebrafish

OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.
Cell Movement ; genetics ; Cytoskeletal Proteins ; genetics ; metabolism ; Cytoskeleton ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; physiology ; Humans ; Neovascularization, Physiologic ; genetics

OBJECTIVE: To explore the interaction between arginine functionalized hydroxyapatite (HAP/Arg) nanoparticles and endothelial cells, and to investigate mechanisms for endocytosis kinetics and endocytosis.
 METHODS: Human umbilical vein endothelial cells (HUVECs) were selected as the research model.Cellular uptake of HAP/Arg nanoparticles were observed by laser scanning confocal microscopy.Average fluorescence intensity of cells after ingestion with different concentrations of HAP/Arg nanoparticles were determined by flow cytometer and atomic force microscopy.
 RESULTS: The HAP/Arg nanoparticles with doped terbium existed in cytoplasm, and most of them distributed around the nucleus area after cellular uptake by HUVECs. Cellular uptake process of HAP/Arg nanoparticles in HUVECs was in a time and concentration dependent manner. 4 h and 50 mg/L was the best condition for uptake. HAP/Arg nanoparticles were easier to be up-taken into the cells than HAP nanoparticles without arginine functionalized.
 CONCLUSION HAP/Arg nanoparticles are internalized by HUVECs cells through an active transport and energy-dependent endocytosis process, and it is up-taken by cells mainly through caveolin-mediated endocytosis, but the clathrin-dependent endocytic pathway is also involved..
Arginine ; pharmacology ; Biological Transport, Active ; physiology ; Caveolins ; physiology ; Cells, Cultured ; Clathrin ; physiology ; Durapatite ; pharmacokinetics ; Endocytosis ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Nanoparticles ; metabolism

BACKGROUNDEndothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells (EPCs) derived from human early fetal aortas in rat injured arteries.

METHODSThe carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas (<14 weeks) were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry.

RESULTSGreen fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control (P < 0.05) and the residual lumen area was larger (P < 0.05). After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human von Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment.

CONCLUSIONEPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.

Animals ; Carotid Arteries ; pathology ; Cell Adhesion ; physiology ; Cell Survival ; physiology ; Cell Transplantation ; Endothelial Progenitor Cells ; cytology ; physiology ; Humans ; Immunohistochemistry ; Microscopy, Fluorescence ; Neointima ; therapy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the effect of colon cancer cell-derived interleukin-1α on the migration and proliferation of human umbilical vein endothelial cells as well as the role of IL-1α and IL-1ra in the angiogenesis process.

METHODSWestern blot was used to detect the expression of IL-1α and IL-1R1 protein in the colon cancer cell lines with different liver metastatic potential. We also examined how IL-1α and IL-1ra influence the proliferation and migration of umbilical vascular endothelial cells assessed by PreMix WST-1 assay and migration assay, respectively. Double layer culture technique was used to detect the effect of IL-1α on the proliferation and migration of vascular endothelial cells and the effect of IL-1ra on the vascular endothelial cells.

RESULTSWestern blot analysis showed that IL-1α protein was only detected in highly metastatic colon cancer HT-29 and WiDr cells, but not in the lowly metastatic CaCo-2 and CoLo320 cells.Migration assay showed that there were significant differences in the number of penetrated cells between the control (17.9±3.6) and 1 ng/ml rIL-1α group (23.2±4.2), 10 ng/ml rIL-1α group (31.7±4.5), and 100 ng/ml rIL-1α group (38.6±4.9), showing that it was positively correlated with the increasing concentration of rIL-1α (P<0.01 for all). The proliferation assay showed that the absorbance values were 1.37±0.18 in the control group, and 1.79±0.14 in the 1 ng/ml rIL-1α group, 2.14±0.17 in the 10 ng/ml rIL-1α group, and 2.21±0.23 in the 100 ng/ml rIL-1α group, showing a positive correlation with the increasing concentration of rIL-1α(P<0.01 for all). IL-1ra significantly inhibited the proliferation and migration of vascular endothelial cells (P<0.01). The levels of VEGF protein were (1.697±0.072) ng/ml, (3.507±0.064)ng/ml and (4.139±0.039)ng/ml in the control, HUVECs+ IL-1α and HUVECs+ HT-29 co-culture system groups, respectively, showing a significant difference between the control and HUVECs+ 10 pg/ml rIL-1α groups and between the control and HUVECs+ HT-29 groups (P<0.01 for both).

CONCLUSIONSOur findings indicate that colon cancer cell-derived IL-1α plays an important role in the liver metastasis of colon cancer through increased VEGF level of the colon cancer cells and enhanced vascular endothelial cells proliferation, migration and angiogenesis, while IL-1ra can suppress the effect of IL-1α and inhibit the angiogenesis in colon cancer.

Blotting, Western ; Caco-2 Cells ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Coculture Techniques ; Colonic Neoplasms ; blood supply ; metabolism ; pathology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Interleukin 1 Receptor Antagonist Protein ; metabolism ; physiology ; Interleukin-1alpha ; metabolism ; physiology ; Liver Neoplasms ; secondary ; Neovascularization, Pathologic ; etiology