Dual drug-loaded polymeric mixed micelles for ovarian cancer: Approach to enhanced therapeutic efficacy of albendazole and paclitaxel.

Chemotherapy resistance remains a significant challenge in treating ovarian cancer effectively. This study addresses this issue by utilizing a dual drug-loaded nanomicelle system comprising albendazole (ABZ) and paclitaxel (PTX), encapsulated in a novel carrier matrix of D-tocopheryl polyethylene glycol 1000 succinate vitamin E (TPGS), soluplus and folic acid. Our objective was to develop and optimize this nanoparticulate delivery system using solvent evaporation techniques to enhance the therapeutic efficacy against ovarian cancer. The formulation process involved pre-formulation, formulation, optimization, and comprehensive characterization of the micelles. Optimization was conducted through a 32 factorial design, focusing on the effects of polymer ratios on particle size, zeta potential, polydispersity index (PDI) and entrapment efficiency (%EE). The optimal formulation demonstrated improved dilution stability, as indicated by a critical micelle concentration (CMC) of 0.0015 mg/mL for the TPGS-folic acid conjugate (TPGS-FOL). Extensive characterization included differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR). The release profile exhibited an initial burst followed by sustained release over 90 h. The cytotoxic potential of the formulated micelles was superior to that of the drugs alone, as assessed by MTT assays on SKOV3 ovarian cell lines. Additionally, in vivo studies confirmed the presence of both drugs in plasma and tumour tissues, suggesting effective targeting and penetration. In conclusion, the developed TPGS-Fol-based nanomicelles for co-delivering ABZ and PTX show promising results in overcoming drug resistance, enhancing solubility, sustaining drug release, and improving therapeutic outcomes in ovarian cancer treatment.

Inhibition of soluble epoxide hydrolase enhances the dentin-pulp complex regeneration mediated by crosstalk between vascular endothelial cells and dental pulp stem cells.

BACKGROUND: Revascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH. METHODS: In vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining. RESULTS: In vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-ß blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment. CONCLUSIONS: TPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-ß. TPPU boosts the angiogenic-odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration.

PDLCs and EPCs Co-Cultured on Ta Discs: A Golden Fleece for "Compromised" Osseointegration.

Material research in tissue engineering forms a vital link between basic cell research and animal research. Periodontal ligament cells (PDLCs, P) from the tooth have an osteogenic effect, whereas endothelial progenitor cells (EPCs, E) assist in neovascularization. In the present study, the interaction of PDLCs and EPCs with Tantalum (Ta, I) discs, either alone (IP or IE group) or in 1:1 (IPE) ratio was explored. Additionally, surface analysis of Ta discs with respect to different types and cycles of sterilization and disinfection procedures was evaluated. It was observed that Ta discs could be used for a maximum of three times, after which the changes in properties of Ta discs were detrimental to cell growth, irrespective of the type of the protocol. Cell-Disc's analysis revealed that cell proliferation in the IE group at day 6 and day 10 was significantly higher (p < 0.05) than other groups. A cell viability assay revealed increased live cells in the IPE group than in the IP or IE group. Similarly, adhesion and penetration of cells in the IPE group were not only higher, but also had an increased thickness of cellular extensions. RT-PCR analysis revealed that on day 8, both osteogenic (ALP, RUNX-2, and BSP) and angiogenic genes (VEGFR-2, CD31) increased significantly in the IPE group as compared to the IP or IE groups (p < 0.05). In conclusion, Ta discs promoted cellular proliferation and increased osteogenic and angiogenic activity by augmenting RUNX-2 and VEGFR-2 activity.

Zoledronate Inhibits Osteoclast Differentiation via Suppressing Vascular Endothelial Growth Factor Receptor 2 Expression.

Bisphosphonate-related osteonecrosis of the jaw (ONJ) is a major oral complication; however, its pathogenesis remains unclear. Impairment of osteoclast differentiation by bisphosphonates may be associated with the pathogenesis of ONJ. In our previous study, we reported that the expression of the gene encoding nuclear factor of activated T cells c1 (NFATc1), a known osteoclast differentiation marker, was significantly silenced by zoledronate, a bisphosphonate, in mouse osteoclast precursor cells (mOCPCs) using cDNA microarray. In the present study, the expression value of the NFATc1 gene was regarded as a cut-off and genes whose expression value was significantly decreased compared with that of the NFATc1 gene were extracted in mOCPCs. For validation, CD11b-positive (CD11b+) cells were used, which were purified from human peripheral blood mononuclear cells as human OCPCs. A total of 19 genes were identified; sequential expression analysis revealed that the gene encoding vascular endothelial growth factor receptor 2 (VEGFR2) was frequently silenced by zoledronate in CD11b+ cells. Furthermore, the number of tartrate-resistant acid phosphatase-positive multinucleated cells was decreased by VEGFR2 suppression using a VEGFR2 neutralizing antibody. Zoledronate inhibits human osteoclast differentiation via suppressing VEGFR2 expression. These results suggest that low expression of VEGFR2 in OCPCs may be involved in the pathogenesis of zoledronate-induced ONJ. The understanding of the role of VEGFR2 on bone remodeling is important to elucidate the pathogenesis of bisphosphonate-related ONJ.

Effects of Strontium-Doped ß-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

It was hypothesized that strontium (Sr)-doped ß-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

Specific Targeting of PEGylated Liposomal Doxorubicin (Doxil®) to Tumour Cells Using a Novel TIMP3 Peptide.

Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers.

MRI analysis of hydrogel-loaded apatinib for local therapy of hepatocellular carcinoma model in nude mice.

AIM: To investigate the effect of local treatment of gadolinium-polyethylene glycol (Gd-PEG) hydrogel containing apatinib injected into hepatocellular carcinoma model of HepG2 in nude mice, and to evaluate the MRI findings in vivo. METHODS: HepG2 cells were treated in vitro and OD 450 value were measured. The four groups (n = 6) were Apatinib-Gd-PEG hydrogel, Gd-PEG hydrogel, Apatinib, and Saline. T1WI and DWI scans were performed before and 1d, 3d, and 14d postoperatively. The samples were examined by histomorphology and immunohistochemistry for CD34 and VEGFR2. Microvessel density (MVD) was evaluated and the average optical density (AOD) of VEGFR2 was obtained by IPP6.0 image software. RESULTS: The OD450-time curves of Gd-PEG hydrogel and phosphate buffer saline (PBS) were similar and that of apatinib at all concentrations are located below; the higher the concentration, the lower the curve. On T1WI and DWI, the newly injected Gd-PEG hydrogel showed significant high signal and was immobilized in the tumor. Subsequently, the size and signal of Gd-PEG hydrogel gradually decreased with time. In Apatinib-Gd-PEG hydrogel group, compared with other three groups, MRI and histomorphology showed that the necrotic area of hepatocellular carcinoma model was larger, immunohistochemistry displayed minimal expression of CD34 and VEGFR2, the AOD of VEGFR2 and MVD differed markedly. CONCLUSION: Gd-PEG hydrogel can significantly enhance and prolong the inhibitory effect of apatinib. It can be visualized by MRI, which can be used to evaluate the local therapeutic effect.

Establishment of tooth blood supply and innervation is developmentally regulated and takes place through differential patterning processes.

Teeth are richly supported by blood vessels and peripheral nerves. The aim of this study was to describe in detail the developmental time-course and localization of blood vessels during early tooth formation and to compare that to innervation, as well as to address the putative role of vascular endothelial growth factor (VEGF), which is an essential regulator of vasculature development, in this process. The localization of blood vessels and neurites was compared using double immunofluorescence staining on sections at consecutive stages of the embryonic (E) and postnatal (PN) mandibular first molar tooth germ (E11-PN7). Cellular mRNA expression domains of VEGF and its signaling receptor VEGFR2 were studied using sectional radioactive in situ hybridization. Expression of VEGF mRNA and the encoded protein were studied by RT-PCR and western blot analysis, respectively, in the cap and early bell stage tooth germs, respectively. VEGFR2 was immunolocalized on tooth tissue sections. Smooth muscle cells were investigated by anti-alpha smooth muscle actin (αSMA) antibodies. VEGF showed developmentally regulated epithelial and mesenchymal mRNA expression domains including the enamel knot signaling centers that correlated with the growth and navigation of the blood vessels expressing Vegfr2 and VEGFR2 to the dental papilla and enamel organ. Developing blood vessels were present in the jaw mesenchyme including the presumptive dental mesenchyme before the appearance of the epithelial dental placode and dental neurites. Similarly, formation of a blood vessel plexus around the bud stage tooth germ and ingrowth of vessels into dental papilla at E14 preceded ingrowth of neurites. Subsequently, pioneer blood vessels in the dental papilla started to receive smooth muscle coverage at the early embryonic bell stage. Establishment and patterning of the blood vessels and nerves during tooth formation are developmentally regulated, stepwise processes that likely involve differential patterning mechanisms. Development of tooth vascular supply is proposed to be regulated by local, tooth-specific regulation by epithelial-mesenchymal tissue interactions and involving tooth target expressed VEGF signaling. Further investigations on tooth vascular development by local VEGF signaling, as well as how tooth innervation and development of blood vessels are integrated with advancing tooth organ formation by local signaling mechanisms, are warranted.

Gene expression of growth factors with angiogenic potential in human dental pulp tissue from teeth with complete and incomplete root development.

AIM: To quantify the expression of angiogenic growth factors (ANG2, VEGFA, TGFß1) and their corresponding receptors (VEGFR1, VGFR2, NRP1 and TGFßR1) in human dental pulps from extracted third molars with complete and incomplete root development. METHODOLOGY: Fifty-six dental pulp samples obtained from freshly extracted human third molars were divided equally into two groups according to their stage of root development; 28 third molars with complete root development and 28 third molars with incomplete root development. All samples were processed and total RNA was extracted, cDNA was then synthetized for each sample and the target genes expression profiles for ANG2, VEGFA, VEGFR1, VEGFR2, NRP1, TGFß1 and TGFßR1 were obtained by RT2-PCR. The data was analysed with a Student's t-test to compare the replicate ∆∆Ct values for each gene. RESULTS: Teeth with incomplete root development were associated with a significantly greater gene expression of TGFßR1 (P = 0.03), whereas in teeth with complete root development the genes that had significantly greater expression were VEGFA (P = 0.04). CONCLUSION: The angiogenic growth factors (ANG2, VEGFA, TGFß1) and their receptors (NRP1, VEGFR1, VEGFR2 and TGFßR1) were expressed in pulps of teeth with complete and incomplete root development measured by RT2-PCR, with TGFBR1 genes being significantly different in teeth with incomplete root development and VEGFA genes in teeth with complete root development.

Vascular endothelial growth factor A/Vascular endothelial growth factor receptor 2 axis promotes human dental pulp stem cell migration via the FAK/PI3K/Akt and p38 MAPK signalling pathways.

AIM: To investigate the effects of vascular endothelial growth factor A (VEGFA) and the underlying molecular mechanisms on the migration of human dental pulp stem cells (hDPSCs). METHODOLOGY: The expression of VEGFA in inflammatory pulp tissue and lipopolysaccharide (LPS)-stimulated dental pulp cells was examined by immunofluorescence staining and qRT-PCR. The migration of hDPSCs was detected using transwell migration and wound healing assays. The activation of FAK, PI3K, Akt and p38 signalling was evaluated by Western blot analysis. Silence RNA (siRNA) technology was utilized to knockdown the expression of VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR). PF573228 (inhibitor of FAK), LY294002 (inhibitor of PI3K), SB203580 (inhibitor of p38) and SU5416 (inhibitor of VEGFR2) were employed to investigate the effect of VEGFA on the migratory mechanism of hDPSCs. Data were analysed statistically using the Student's t-test or one-way ANOVA. RESULTS: The expression levels of VEGFA in inflammatory pulp tissue in vivo and LPS-stimulated dental pulp cells in vitro were significantly greater than those in the control groups (P < 0.05). Vascular endothelial growth factor A promoted the migration of hDPSCs in a concentration-dependent manner. Several signalling pathways, including FAK, PI3K, Akt and p38, were activated by VEGFA in a dose- and time-dependent manner in hDPSCs. The VEGFA-induced migration of hDPSCs was significantly inhibited with drug inhibitors such as PF573228, LY294002, SB203580 or SU5416 (P < 0.05). These signalling pathways activated by VEGFA stimulation were significantly suppressed by pre-treatment with inhibitor of VEGFR2 (SU5416) or transfection with siRNA of VRGFR2 (P < 0.05) but not VEGFR1 siRNA. CONCLUSIONS: Vascular endothelial growth factor A/VEGFR2 axis promoted the migration of hDPSCs via the FAK/PI3K/Akt and p38 MAPK signalling pathways. These findings reveal a novel molecular mechanism for cell migration of hDPSCs, which may contribute to the remodelling of pulp tissue and dentine.

VEGF- and VEGFR2-Targeted Liposomes for Cisplatin Delivery to Glioma Cells.

Targeted delivery of anticancer drugs to brain tumors, especially glioblastoma multiforme, which is the most frequent and aggressive type, is one of the important objectives in nanomedicine. Vascular endothelial growth factor (VEGF) and its receptor type II (VEGFR2) are promising targets because they are overexpressed by not only core tumor cells but also by migrated glioma cells, which are responsible for resistance and rapid progression of brain tumors. The purpose of the present study was to develop the liposomal drug delivery system combining enhanced loading capacity of cisplatin and high binding affinity to glioma cells. This was achieved by using of highly soluble cisplatin analogue, cis-diamminedinitratoplatinum(II), and antibodies against the native form of VEGF or VEGFR2 conjugated to liposome surface. The developed drug delivery system revealed sustained drug release profile, high affinity to antigens, and increased uptake by glioma C6 and U-87 MG cells. Pharmacokinetic study on glioma C6-bearing rats revealed prolonged blood circulation time of the liposomal formulation. The above features enabled the present drug delivery system to overcome both poor pharmacokinetics typical for platinum formulations and low loading capacity typical for conventional liposomal cisplatin formulations.

Internalization of Vectored Liposomes in a Culture of Poorly Differentiated Tumor Cells.

Internalization of liposomal nanocontainers conjugated with monoclonal antibodies to VEGF, VEGFR2 (KDR), and proteins overproduced in the tumor tissue was studied in vitro on cultures of poorly differentiated tumor cells. Comparative analysis of accumulation of vectored liposomes in the tumor cells was performed by evaluating co-localization of labeled containers and cell organelles by laser scanning confocal microscopy. We observed nearly 2 times more active penetration and accumulation of liposomes vectored with antibodies in the tumor cells in comparison with non-vectored liposomes. Selective clathrin-dependent penetration of vectored liposomes into tumor cells was demonstrated by using pharmacological agents inhibiting endocytosis.

Use of capillary electrophoresis as a versatile tool to measure interaction constants between a KDR-binding PEGylated lipopeptide and pegylated phospholipid micelles.

In the frame of our molecular imaging activities, a PEGylated lipopeptide has been developed as a specific ligand for the human vascular endothelial growth factor receptor 2, which is considered as one of the important molecular marker of angiogenesis. In this study, the potential of affinity capillary electrophoresis (ACE) is evaluated to measure the interactions of an active PEGylated lipopeptide, its hydrolysis product and its precursor consisting of a peptide structure with different micelles including Brij-35, Tween-20, and pegylated phospholipids. Given the amphiphilic structure of the PEGylated lipopeptide, a MEKC method allowing the simultaneous separation of the compounds of interest was set up, using low percentages of acetonitrile. Analytes were resolved using a BGE consisting of 100 mM borate buffer pH 9.0, 1 mM Brij, and 25% acetonitrile. Optimized conditions were then used to perform ACE experiments. The affinity constants of the analytes with the micelles were calculated on the basis of their mobility decrease when surfactant concentration increased in the electrolyte. The use of different linearization models to estimate affinity constants was discussed and comparison of different surfactants was reported. PEGylated lipopeptide interacted more strongly with pegylated phospholipid micelles than with Brij-35 or Tween-20. Moreover, it is likely that the chemical structure of the compounds, and particularly the lipidic part of the molecules, significantly affects the interaction with micelles. In conclusion, the ACE method can be readily applied to investigate interactions of our targeting lipopeptides with various micelles currently used for the preparation of pharmaceutical vehicles.

Potent anti-angiogenic component in Kaempferia galanga L. and its mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the roots of Kaempferia galanga has been used to treat high blood pressure, chest pain, headache, toothache, rheumatism, indigestion, cough, inflammation and cancer in Asia. Nevertheless, most of its pharmacological studies were focused on ethanolic extracts and volatile oils. The exact active chemical constituents and their underlying mechanisms are still poorly understood, especially towards its anti-cancer treatment. Inhibition of angiogenesis is an important atrategy to inhibit tumor growth. It has been reported that the low polar component of the plant possessed anti-angiogenic activity. Yet, the potent compound which is responsible for the effect and its molecular mechanism has not been reported. AIM OF THE STUDY: To determine the potent anti-angiogenic component in K.galanga and its mechanism of action. MATERIAL AND METHODS: The low polar components of the plant were concentrated using the methods of supercritical fluid extraction (SFE), subcritical extraction (SCE) and steam distillation (SD). The anti-angiogenic activity of the three extracts was evaluated using a zebrafish model. The content of the active compound in those extracts was determined with HPLC analysis. The in-vitro and in-vivo activity of the isolated compound was evaluated using human umbilical vein endothelial cells (HUVECs) model, the aortic ring assay and the matrigel plug assay, respectively. Its molecular mechanism was further studied by the western blotting assay and computer-docking experiments. Besides, its cytotoxicity on cancer and normal cell lines was evaluated using the cell-counting kit. RESULTS: HPLC results showed that trans-ethyl p-methoxycinnamate (TEM) was the major component of the extracts. The extract of SFE showed the best effect as it has the highest content of TEM. TEM could inhibit vascular endothelial growth factor (VEGF)-induced viability, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Mechanistic study showed that it could suppress tyrosine kinase activity of the receptor of VEGF (VEGFR2) and alter its downstream signaling pathways. In addition, the molecular docking showed that the binding of TEM and VEGFR2 is stable, which mainly attributed to the non-covalent binding interaction. Beside, TEM possessed little toxicity to both cancer and normal cells. CONCLUSION: TEM is the major anti-angiogenic component present in K. galanga and its anti-angiogenic property rather than toxicity provides scientific basis for the traditional use of K. galanga in cancer treatment.

The Impact of the VEGF/VEGFR2/PI3K/AKT Signaling Axis on the Proliferation and Migration Abilities of Human Dental Pulp Stem Cells.

The potential therapeutic benefits of human dental pulp stem cells (HDPSCs) in dental regenerative medicine have been demonstrated. However, little is known about the molecular mechanisms regulating the biological characteristics of HDPSCs. The experiment aims to explore whether VEGF activates signaling pathways such as FAK, PI3K, Akt, and p38 in HDPSCs, and to investigate the molecular mechanisms by which VEGF influences proliferation and migration of HDPSCs. Normal and inflamed human dental pulp (HDP) samples were collected, and the levels of VEGF in HDP were assessed. HDPSCs were cultured and purified. HDPSCs were stimulated with lipopolysaccharide (LPS) at gradient concentrations, and real-time quantitative polymerase chain reaction (qPCR) was used to assess changes in VEGF mRNA. Gradient concentrations of VEGF were used to stimulate HDPSCs, and cell migration ability was evaluated through scratch assays and Transwell chamber experiments. Phosphorylation levels of FAK, AKT, and P38 were assessed using Western blotting. Inhibitors of VEGFR2, FAK, AKT, P38, and VEGF were separately applied to HDPSCs, and cell migration ability and phosphorylation levels of FAK, AKT, and P38 were determined. The results indicated significant differences in VEGF levels between normal and inflamed HDP tissues, with levels in the inflamed state reaching 435% of normal levels (normal: 87.91 ng/mL, inflamed: 382.76 ng/mL, P < 0.05). LPS stimulation of HDPSCs showed a significant increase in VEGF mRNA expression with increasing LPS concentrations (LPS concentrations of 0.01, 0.1, 1, and 10 µg/mL resulted in VEGF mRNA expressions of 181.2%, 274.2%, 345.8%, and 460.9%, respectively, P < 0.05). VEGF treatment significantly enhanced the migration ability of HDPSCs in Transwell chamber experiments, with migration rates increasing with VEGF concentrations (VEGF concentrations of 0, 1, 10, 20, 50, and 100 ng/mL resulted in migration rates of 8.41%, 9.34%, 21.33%, 28.41%, 42.87%, and 63.15%, respectively, P < 0.05). Inhibitors of VEGFR2, FAK, AKT, P38, and combined VEGF stimulation demonstrated significant migration inhibition, with migration rates decreasing to 8.31%, 12.64%, 13.43%, 18.32%, and 74.17%, respectively. The migration rate with combined VEGF stimulation showed a significant difference (P < 0.05). The analysis of phosphorylation levels revealed that VEGF stimulation significantly activated phosphorylation of FAK, AKT, and P38, with phosphorylation levels increasing with VEGF concentrations (P < 0.05). The VEGF/VEGFR2 signaling axis regulated the migration ability of HDPSCs through the FAK/PI3K/AKT and P38MAPK pathways. This finding highlighted not only the crucial role of VEGF in injury repair of HDPSCs but also provided important clues for a comprehensive understanding of the potential applications of this signaling axis in dental regenerative medicine.

Immunohistochemical characterization of stem cell, vascular, neural, and differentiation markers in the apical papilla and dental pulp of human teeth at various stages of root development.

This study aimed to evaluate the expression of several differentiation markers in the apical papilla (AP) and dental pulp (DP) of human permanent teeth. Twenty young human teeth were extracted and classified according to three Moorrees tooth development stages: initial root formation (Ri), root length ½ (R1/2), and root length complete (Rc). Immunohistochemical assays were performed using STRO-1, VEGF Receptor-2, Neurofilament heavy (NFH), and Nestin antibodies and analyzed under light microscopy. Decalcified, formalin fixed paraffin embedded tooth sections stained with hematoxylin and eosin showed an apical cell rich zone between the DP and AP. The AP revealed fewer vascular and cellular components than the DP. STRO-1 was expressed on vascular and neuronal elements beneath the odontoblast (OB) and in the sub-odontoblastic (SOB) zone, and VEGFR-2 positive cells were observed in the endothelium, arterioles, and blood vessels. Neuroepithelial stem cell protein (Nestin) was highly expressed in differentiated odontoblasts in the predentin odontotoblast and odontoblast cell processes. Neurofilament heavy (NFH) was expressed in mature axons throughout the DP. STRO-1 and VEGFR-2 microvascular expression was higher at the stages Ri and R1/2 while STRO-1 and NFH expression showed strong spatial distribution of Rc neuronal elements as compared to Ri and R1/2. Differentiated OB and SOB cells showed Nestin expression, indicating a reservoir of newly differentiated odontoblast-like cells.

Ramucirumab-related Oral Pyogenic Granuloma: A Report of Two Cases.

Pyogenic granuloma (PG) is a granulomatous elevated lesion that occurs on the skin and mucous membranes. We herein report two cases of intra-oral PG that developed during the administration of ramucirumab for gastric cancer. Case 1 involved a 55-year-old man with a 6-mm tumor on the right tongue, and case 2 involved a 67-year-old man with a 5-mm tumor on the upper lip. The imbalance in angiogenesis caused by ramucirumab and the deterioration in the local oral environment were suggested to have caused the PG. Medical and dental collaboration is essential during the administration of ramucirumab.

Silencing VEGFR-2 Hampers Odontoblastic Differentiation of Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Functionalization with a VEGFR2-binding antibody fragment leads to enhanced endothelialization of a cardiovascular stent in vitro and in vivo.

Rapid endothelialization of cardiovascular stents is critical to prevent major clinical complications such as restenosis. Reconstruction of the native endothelium on the stent surface can be achieved by the capture of endothelial progenitor cells (EPCs) or neighboring endothelial cells (ECs) in vivo. In this study, stainless steel cardiovascular stents were functionalized with recombinant scFv antibody fragments specific for vascular endothelial growth factor receptor-2 (VEGFR2) that is expressed on EPCs and ECs. Anti-VEGFR2 scFvs were expressed in glycosylated form in Escherichia coli and covalently attached to amine-functionalized, titania-coated steel disks and stents. ScFv-coated surfaces exhibited no detectable cytotoxicity to human ECs or erythrocytes in vitro and bound 15 times more VEGFR2-positive human umbilical vein ECs than controls after as little as 3 min. Porcine coronary arteries were successfully stented with scFv-coated stents with no adverse clinical events after 30 days. Endovascular imaging and histology revealed coverage of the anti-VEGFR2 scFv-coated stent with a cell layer after 5 days and the presence of a neointima layer with a minimum thickness of 80 µm after 30 days. Biofunctionalization of cardiovascular stents with endothelial cell-capturing antibody fragments in this manner offers promise in accelerating stent endothelialization in vivo. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:213-224, 2020.

A Dual Receptor Targeting- and BBB Penetrating- Peptide Functionalized Polyethyleneimine Nanocomplex for Secretory Endostatin Gene Delivery to Malignant Glioma.

PURPOSE: Vascular endothelial growth factor receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1) are two prominent synergistic receptors overexpressed on new blood vessels in glioma and may be promising targets for antiglioma therapy. The aim of this study was to design a dual receptor targeting and blood-brain barrier (BBB) penetrating peptide-modified polyethyleneimine (PEI) nanocomplex that can efficiently deliver the angiogenesis-inhibiting secretory endostatin gene (pVAXI-En) to treat glioma. MATERIALS AND METHODS: We first constructed the tandem peptide TAT-AT7 by conjugating AT7 to TAT and evaluated its binding affinity to VEGFR-2 and NRP-1, vasculature-targeting ability and BBB crossing capacity. Then, TAT-AT7-modified PEI polymer (PPTA) was synthesized, and a pVAXI-En-loaded PPTA nanocomplex (PPTA/pVAXI-En) was prepared. The physicochemical properties, cytotoxicity, transfection efficiency, capacities to cross the BBB and BTB (blood-tumor barrier) and glioma-targeting properties of PPTA/pVAXI-En were investigated. Moreover, the in vivo anti-angiogenic behaviors and anti-glioma effects of PPTA/pVAXI-En were evaluated in nude mice. RESULTS: The binding affinity of TAT-AT7 to VEGFR-2 and NRP-1 was approximately 3 to 10 times greater than that of AT7 or TAT. The cellular uptake of TAT-AT7 in endothelial cells was 5-fold and 119-fold greater than that of TAT and AT7 alone, respectively. TAT-AT7 also displayed remarkable efficiency in penetrating the BBB and glioma tissue in vivo. PPTA/pVAXI-En exhibited lower cytotoxicity, stronger BBB and BTB traversing abilities, higher selective glioma targeting and better gene transfection efficiency than PEI/pVAXI-En. More importantly, PPTA/pVAXI-En significantly suppressed the tube formation and migration of endothelial cells, inhibited glioma growth, and reduced the microvasculature in orthotopic U87 glioma-bearing nude mice. CONCLUSION: Our study demonstrates that PPTA/pVAXI-En can be exploited as an efficient dual-targeting nanocomplex to cross the BBB and BTB, and hence it represents a feasible and promising nonviral gene delivery system for effective glioma therapy.