Increased Blood Vessel Formation Research Articles

Evaluating the Healing Properties of Quercetin-Enhanced Nanoparticle Ointments in Diabetic Rats

Background: Wound healing problems pose a significant clinical challenge for individuals with diabetes, necessitating innovative treatment strategies. Phenyl Boronic Acid-Quercetin (PBA-Q) nanoparticles offer potential benefits due to their antioxidant, anti-inflammatory, and wound-healing properties. Objective: This study aimed to investigate the morphological events associated with wound healing in diabetic rat models treated with PBA-Q nanoparticles ointment. Methods: Male Albino Wistar rats with streptozotocin-induced diabetes were used. Diabetes was confirmed with blood glucose levels exceeding 250 mg/dL. Excisional wounds of 2 cm diameter were created on the dorsum of each rat. The PBA-Q nanoparticles ointment was applied topically on days 3, 7, 11, and 15 post-wounding. Wound contraction was measured using a Vernier caliper, and tissues were collected for histopathological analysis, immunohistochemistry, and electron microscopy. Statistical analysis was performed using SPSS version 25.0. Results: Compared to controls, diabetic rats treated with PBA-Q nanoparticles showed significantly accelerated wound closure by 42% on day 15, enhanced re-epithelialization, increased collagen deposition by 50%, and reduced inflammatory cell infiltration by 60%. Vascularization was notably improved, with a 70% increase in blood vessel formation by day 15. Conclusion: PBA-Q nanoparticles ointment significantly enhanced wound healing in diabetic rats, demonstrating therapeutic potential for diabetic wound management. Further research is needed to validate these findings in human clinical trials.

Electroacupuncture facilitates vascular normalization by inhibiting Glyoxalase1 in endothelial cells to attenuate glycolysis and angiogenesis in triple-negative breast cancer

Recent therapeutic strategies for the treatment of triple-negative breast cancer (TNBC) have shifted the focus from vascular growth factors to endothelial cell metabolism. This study highlights the underexplored therapeutic potential of peri-tumoral electroacupuncture, a globally accepted non-pharmacological intervention for TNBC, and molecular mechanisms. Our study showed that peri-tumoral electroacupuncture effectively reduced the density of microvasculature and enhanced vascular functionality in 4T1 breast cancer xenografts, with optimal effects on day 3 post-acupuncture. The timely integration of peri-tumoral electroacupuncture amplified the anti-tumor efficacy of paclitaxel. Multi-omics analysis revealed Glyoxalase 1 (Glo1) and the associated methylglyoxal-glycolytic pathway as key mediators of electroacupuncture-induced vascular normalization. Peri-tumoral electroacupuncture notably reduced Glo1 expression in the endothelial cells of 4T1 xenografts. Using an in vivo matrigel plug angiogenesis assay, we demonstrated that either Glo1 knockdown or electroacupuncture inhibited angiogenesis. In contrast, Glo1 overexpression increased blood vessel formation. In vitro pharmacological inhibition and genetic knockdown of Glo1 in human umbilical vein endothelial cells inhibited proliferation and promoted apoptosis via downregulating the methylglyoxal-glycolytic pathway. The study using the Glo1-silenced zebrafish model further supported the role of Glo1 in vascular development. This study underscores the pivotal role of Glo1 in peri-tumoral electroacupuncture, spotlighting a promising avenue for enhancing vascular normalization and improving TNBC treatment outcomes.

Photobiomodulation therapy’s impact on angiogenesis and osteogenesis in orthodontic tooth movement: in vitro and in vivo study

BackgroundThis study explores the effectiveness of Photobiomodulation Therapy (PBMT) in enhancing orthodontic tooth movement (OTM), osteogenesis, and angiogenesis through a comprehensive series of in vitro and in vivo investigations. The in vitro experiments involved co-culturing MC3T3-E1 and HUVEC cells to assess PBMT’s impact on cell proliferation, osteogenesis, angiogenesis, and associated gene expression. Simultaneously, an in vivo experiment utilized an OTM rat model subjected to laser irradiation at specific energy densities.MethodsIn vitro experiments involved co-culturing MC3T3-E1 and HUVEC cells treated with PBMT, enabling a comprehensive assessment of cell proliferation, osteogenesis, angiogenesis, and gene expression. In vivo, an OTM rat model was subjected to laser irradiation at specified energy densities. Statistical analyses were performed to evaluate the significance of observed differences.ResultsThe results revealed a significant increase in blood vessel formation and new bone generation within the PBMT-treated group compared to the control group. In vitro, PBMT demonstrated positive effects on cell proliferation, osteogenesis, angiogenesis, and gene expression in the co-culture model. In vivo, laser irradiation at specific energy densities significantly enhanced OTM, angiogenesis, and osteogenesis.ConclusionsThis study highlights the substantial potential of PBMT in improving post-orthodontic bone quality. The observed enhancements in angiogenesis and osteogenesis suggest a pivotal role for PBMT in optimizing treatment outcomes in orthodontic practices. The findings position PBMT as a promising therapeutic intervention that could be seamlessly integrated into orthodontic protocols, offering a novel dimension to enhance overall treatment efficacy. Beyond the laboratory, these results suggest practical significance for PBMT in clinical scenarios, emphasizing its potential to contribute to the advancement of orthodontic treatments. Further exploration of PBMT in orthodontic practices is warranted to unlock its full therapeutic potential.

The osteogenic and angiogenic potential of microRNA-26a delivered via a non-viral delivery peptide for bone repair

Bone-related injuries and diseases are among the most common causes of morbidity worldwide. Current bone-regenerative strategies such as auto- and allografts are invasive by nature, with adverse effects such as pain, infection and donor site morbidity. MicroRNA (miRNA) gene therapy has emerged as a promising area of research, with miRNAs capable of regulating multiple gene pathways simultaneously through the repression of post-transcriptional mRNAs. miR-26a is a key regulator of osteogenesis and has been found to be upregulated following bone injury, where it induces osteodifferentiation of mesenchymal stem cells (MSCs) and facilitates bone formation. This study demonstrates, for the first time, that the amphipathic, cell-penetrating peptide RALA can efficiently deliver miR-26a to MSCs in vitro to regulate osteogenic signalling. Transfection with miR-26a significantly increased expression of osteogenic and angiogenic markers at both gene and protein level. Using a rat calvarial defect model with a critical size defect, RALA/miR-26a NPs were delivered via an injectable, thermo-responsive Cs-g-PNIPAAm hydrogel to assess the impact on both rate and quality of bone healing. Critical defects treated with the RALA/miR-26a nanoparticles (NPs) had significantly increased bone volume and bone mineral density at 8 weeks, with increased blood vessel formation and mechanical properties. This study highlights the utility of RALA to deliver miR-26a for the purpose of bone healing within an injectable biomaterial, warranting further investigation of dose-related efficacy of the therapeutic across a range of in vivo models.

Evaluation of Mast Cell Density in Mucoepidermoid Carcinoma and Pleomorphic Adenoma.

Mast cells are round to elliptical cells that originate from bone marrow stem cells and enter the peripheral blood. By releasing inflammatory mediators, these cells are involved in type I hypersensitivity, wound healing, defense against pathogens, increased blood vessel formation, and destruction of the extracellular matrix. There are contradictory results regarding the role of mast cells in tumor lesions. Considering the contradictory results and few studies on the density of mast cells in salivary tumors, the present study investigated and compared the density of mast cells in two common salivary gland tumors. In the cross-sectional study after reviewing the records of patients referred to the Pathology Department of the School of Dentistry and Shahid Sadoughi Hospital in Yazd, 15 blocks of each of the mucoepidermoid carcinoma and pleomorphic adenoma tumors were taken. After Giemsa staining of the samples, the average of stained cells in 10 random fields under 400× magnification was counted. The results were analyzed using statistical tests of t-test, ANOVA, Chi-square, and Mann-Whitney in SPSS ver. 22. The average mast cell counts in pleomorphic adenoma (4.2) was higher than muco-epidermoid carcinoma (1.7) but there was no significant relationship (p= 0.305). In mucoepidermoid carcinoma, the numbers of mast cells increased with increasing tumor grade (low: 0/467 moderate: 1/567 high: 2/983) and there was a significant relationship (p= 0.009). According to the results of the present study, it seems that the mast cells accumulation may be secondarily associated with inflammatory responses due to cell accumulation and tissue destruction by tumor cells.

VEGF-Encoding, Gene-Activated Collagen-Based Matrices Promote Blood Vessel Formation and Improved Wound Repair

Disruption in vascularization during wound repair can severely impair healing. Proangiogenic growth factor therapies have shown great healing potential; however, controlling growth factor activity and cellular behavior over desired healing time scales remains challenging. In this study, we evaluated collagen-mimetic peptide (CMP) tethers for their capacity to control growth factor gene transfer and growth factor activity using our recently developed gene-activated hyaluronic acid-collagen matrix (GAHCM). GAHCM was comprised of DNA/polyethyleneimine (PEI) polyplexes that were retained on hyaluronic acid (HA)-collagen hydrogels using CMPs. We hypothesized that using CMP-collagen tethers to control vascular endothelial growth factor-A (VEGF-A) gene delivery in fibroblasts would provide a powerful strategy to modulate the proangiogenic behaviors of endothelial cells (ECs) for blood vessel formation, resulting in enhanced wound repair. In co-culture experiments, we observed that CMP-modified GAHCM induced tunable gene delivery in fibroblasts as predicted, and correspondingly, VEGF-A produced by the fibroblasts led to increased growth and persistent migration of ECs for at least 7 days, as compared to non-CMP-modified GAHCM. Moreover, when ECs were exposed to fibroblast-containing VEGF-GAHCM with higher levels of CMP modification (50% CMP-PEI, or 50 CP), high CD31 expression was stimulated, resulting in the formation of an interconnected EC network with a significantly higher network volume and a larger diameter network structure than controls. Application of VEGF-GAHCM with 50 CP in murine splinted excisional wounds facilitated prolonged prohealing and proangiogenic responses resulting in increased blood vessel formation, improved granulation tissue formation, faster re-epithelialization, and overall enhanced repair. These findings suggest the benefits of CMP-collagen tethers as useful tools to control gene transfer and growth factor activity for improved treatment of wounds.

Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration.

Healthy regeneration of tissue relies on a well-orchestrated release of growth factors. Herein, we show the use of synthetic glycosaminoglycans for controlled binding and release of growth factors to induce a desired cellular response. First, we screened glycosaminoglycans with growth factors of interest to determine kon (association rate constant), koff (dissociation rate constant), and Kd (equilibrium rate constant). As proof-of-concept, we functionalized an elastin-like recombinamer (ELR) hydrogel with a synthetic glycosaminoglycan and immobilized fibroblast growth factor 2 (FGF2), demonstrating that human umbilical vein endothelial cells culturedon topof ELR hydrogel differentiated into tube-like structures. Taking this concept further, we developed a tunable macroporous ELR cryogel material, containing a synthetic glycosaminoglycan and FGF2 that showed increased blood vessel formation and reduced immune response compared to control when implanted in a subcutaneous mouse model. These results demonstrated the possibility for specific release of desired growth factors in/from a modular 3D scaffold in vitro and in vivo.

Exosomal lncRNA KLF3-AS1 derived from bone marrow mesenchymal stem cells stimulates angiogenesis to promote diabetic cutaneous wound healing

AimsWe focused on BMSC-derived exosomal lncRNA KLF3-AS1 and its significance in diabetic cutaneous wound healing. MethodsPotential interaction between KLF3-AS1 and miR-383, miR-383 and VEGFA were predicted using bioinformatic analysis and validated by luciferase reporter, RIP, and FISH assays. The proliferation, apoptosis, migration and tube formation of HUVECs were evaluated by CCK-8, flow cytometry, wound healing, and tube formation assays, respectively. A murine diabetic cutaneous wound model was used to investigate therapeutic effects of exosomal KLF3-AS1 in vivo. Histological alterations in skin tissues were examined using HE, Masson staining, and immunostaining of CD31. ResultsBMSC-derived exosomal KLF3-AS1 sufficiently promoted proliferation, migration, and tube formation, while inhibited apoptosis of HUVECs challenged by high glucose. The protective effects of exosomal KLF3-AS1 were achieved at least partially by down-regulating miR-383, and boosting the expression of its target, VEGFA. In vivo, exosomes from KLF3-AS1-expressing BMSCs demonstrated the best effects in promoting cutaneous wound healing in diabetic mice, which were associated with minimal weight loss, increased blood vessel formation, reduced inflammation, decreased miR-383 expression, and up-regulated VEGFA. ConclusionsExosomal lncRNA KLF3-AS1 derived from BMSCs induces angiogenesis to promote diabetic cutaneous wound healing.

THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

Background: BM-MSCs contribute to Helicobacter pylori (H. pylori)-induced gastric cancer, but their mechanism is still unclear. The aim of our study was to investigate the specific role and mechanism of BM-MSCs in H. pylori-induced gastric cancer.Main methods: Mice received total bone marrow transplants and were then infected with H. pylori. BM-MSCs were extracted and transplanted into the gastric serosal layer of mice chronically infected with H. pylori. Hematoxylin and eosin staining, immunohistochemistry staining and immunofluorescence were performed to detect tumor growth and angiogenesis in mouse stomach tissues. Chicken chorioallantoic membrane assays, xenograft tumor models, and human umbilical vein endothelial cell tube formation assays were used for in vivo and in vitro angiogenesis studies. THBS4 was screened from RNA-seq analysis of gastric tissues of BM-MSCs transplanted into H. pylori-infected mice.Results: BM-MSCs can migrate to the site of chronic mucosal injury and promote tumor angiogenesis associated with chronic H. pylori infection. Migration of BM-MSCs to the site of chronic mucosal injury induced the upregulation of THBS4, which was also evident in human gastric cancer and correlated with increased blood vessel formation and worse outcome. The THBS4/integrin α2 axis promoted angiogenesis by facilitating the PI3K/AKT pathway in endothelial cells.Conclusions: Our results revealed a novel proangiogenic effect of BM-MSCs in the chronic H. pylori infection microenvironment, primarily mediated by the THBS4/integrin α2 axis, which activates the PI3K/AKT pathway in endothelial cells and eventually induces the formation of new tumor vessels.

Proangiogenic Collagen-Binding Glycan Therapeutic Promotes Endothelial Cell Angiogenesis.

Stimulating angiogenesis during wound healing continues to present a significant clinical challenge, given the limitations of current strategies to maintain therapeutic doses of growth factors and endothelial cell efficacy. Incorporating a balance of specific cues to encourage endothelial cell engraftment and cytokines to facilitate angiogenesis is necessary for blood vessel growth in the proinflammatory wound environment. Here, we incorporate a previously designed peptide (LXW7) capable of binding to the αvβ3 integrin of endothelial cells with a dermatan sulfate glycosaminoglycan backbone grafted with collagen-binding peptides (SILY). By exploiting αvβ3 integrin-mediated VEGF signaling, we propose an alternative strategy to overcome shortcomings of traditional growth factor therapy while homing the peptide to the wound bed. In this study, we describe the synthesis and optimization of LXW7-DS-SILY (LDS) variants and evaluate their angiogenic potential in vitro and in vivo. LDS displayed binding to collagen and endothelial cells. In vitro, the LDS variant with six LXW7 peptides increased endothelial cell proliferation, migration, and tubule formation through increased VEGFR2 phosphorylation compared to nontreated controls. In an in vivo chick chorioallantoic membrane assay, LDS laden collagen hydrogels increased blood vessel formation by 43% in comparison to the organism matched blank hydrogels. Overall, these findings demonstrate the potential of a robust targeted glycan therapeutic for promoting angiogenesis during wound healing.

Proangiogenic Collagen Binding Glycan Therapeutic Promotes Endothelial Cell Health: Potential Application for the Treatment of Ischemic Wounds

Peripheral artery disease and endothelial cell dysfunction due to diabetes contribute to impaired neovascularization and poor healing of ischemic wounds. Treatments addressing this underlying ischemia that remain effective in the highly proteolytic diabetic wound environment are urgently needed to increase the rate of wound healing and reduce diabetes-related lower-limb amputations. Our lab has previously designed a collagen-targeted glycan therapeutic (DS-SILY) capable of protecting collagen-based scaffolds from matrix metalloproteinase (MMP) mediated degradation. Building upon this targeted technology, we designed the next generation glycan therapy, termed LXW7-DS-SILY (LDS), to also contain proangiogenic capabilities. By exploiting αvβ3 integrin-mediated VEGF signaling using our previously identified αvβ3 integrin targeted peptide (LXW7), we propose an alternative strategy to overcome shortcomings of traditional growth factor therapy. In this study, we describe the synthesis and optimization of LDS variants and evaluate their angiogenic potential in vitro and in vivo. LDS displayed binding to collagen and endothelial cells. In vitro, the LDS variant with 6 LXW7 peptides increased endothelial cell proliferation, migration, and tubule formation through increased VEGFR2 phosphorylation compared to non-treated controls. In vivo in a chick chorioallantoic membrane (CAM) assay, LDS laden collagen hydrogels increased blood vessel formation by 43% in comparison to organism matched blank hydrogels. Overall, these findings demonstrate the potential of a robust proangiogenic targeted therapeutic for the treatment of ischemic diabetic wounds.

Poly(N-isopropylacrylamide)-based dual-crosslinking biohybrid injectable hydrogels for vascularization

Injectable hydrogels provide a powerful and non-invasive approach for numerous applications in cell transplantation, growth factor delivery, tissue regeneration and so forth. The properties of injectable hydrogels should be well-tuned for specific applications, where their overall design should ensure biocompatibility, non-toxicity, robust mechanical properties, and most importantly the ability to promote vascularization and integration with the host tissue/organ. Among these criteria, vascularization remains a key design element in the development of functional therapeutic hydrogels for successful translation into clinical settings. To that end, there is still a critical need for the development of the next generation of injectable hydrogels with precisely tuned biophysical and biochemical properties which could simultaneously promote tissue vascularization. In this work, we developed a temperature responsive, dual-crosslinking, biohybrid hydrogels, modified with a vasculogenic peptide for applications in regenerative medicine, specifically tissue vascularization. The synthesized hydrogels consisted of poly(N-isopropylacrylamide)-based copolymer, functionalized gelation and angiogenic VEGF-mimetic QK peptide with enhanced shear-thinning and injectability properties. QK peptide is a VEGF-mimetic vasculogenic peptide which binds to VEGF receptors and activates intercellular pathway for vascularization. Apart from the presence of QK peptide, the mechanical properties of the hydrogels were precisely tuned by altering the polymer concentration, enabling successful assembly and endothelial cell network formation. Extended in vitro studies demonstrated successful encapsulation and homogeneous distribution of endothelial cells within the three-dimensional (3D) environment of the hydrogel matrix with significantly enhanced vascularization in presence of the QK peptide as early as 3 days of culture. A small, preliminary in vivo study in mice showed a trend of increased blood vessel formation in hydrogels that incorporated the QK peptide. Overall, our study presents the design and characterization of injectable, dual-crosslinking and vasculogenic hydrogels with controlled properties which could be utilized for numerous applications in regenerative medicine, minimally invasive cell and drug delivery as well as fundamental studies on tissue vascularization and angiogenesis. Statement of SignificanceIn this work, we synthesized a new class of temperature responsive, dual-crosslinking, biohybrid injectable hydrogels with enhanced vascularization properties for broad applications in regenerative medicine and minimally invasive cell/drug delivery. The developed hydrogels properly accommodated 3D culture, assembly and network formation of endothelial cells, as evidenced by in vitro and in vivo studies.

Injectable nanoclay gels for angiogenesis

The retention and sustained activity of therapeutic proteins at delivery sites are goals of regenerative medicine. Vascular endothelial growth factor (VEGF) has significant potential in promoting the growth and regeneration of blood vessels but is intrinsically labile. This is exacerbated by the inflammatory microenvironments at sites requiring regeneration. For VEGF to be efficacious, it may require a carrier that stabilises it, protects it from degradation and retains it at the site of interest. In this study, we tested the hypothesis that injectable nanoclay gels comprising Laponite™ XLG (a synthetic hectorite clay) can stabilise VEGF and retain it in the active form for therapeutic delivery. To achieve this, VEGF was incorporated in Laponite gels and its activity tested at a range of concentrations using in vitro cell culture tubulogenesis assays and in vivo angiogenesis assays. We found that VEGF-Laponite gels enhanced tubulogenesis in a dose-dependent manner in vitro. When administered subcutaneously in vivo, Laponite was retained at the injection site for up to a period of three weeks and promoted a 4-fold increase in blood vessel formation compared with that of alginate or vehicle controls as confirmed by CD31 staining. Notably, as compared to alginate, Laponite gels did not release VEGF, indicating a strong interaction between the growth factor and the nanoclay and suggesting that Laponite enhancement of VEGF efficacy is due to its retention at the implantation site for a prolonged period. Our approach provides a robust method for the delivery of bioactive recombinant VEGF without the necessity for complex hydrogel or protein engineering. Statement of significanceIn medicine, it is important to deliver drugs to a particular location in the body. Often, however, the drugs are quickly broken down and carried away in the blood before they can exert their effect. In this study, we used a type of synthetic clay, called Laponite™, to preserve a molecule, named VEGF, that stimulates the growth of blood vessels. Previously, we have been able to bind VEGF to the surface of clays, but the clay is not effective when injected or applied as a gel. Herein, we show that we can mix VEGF with the clay and that it strongly stimulates blood vessel growth. We speculate that this would be a useful material for skin wound healing.

Invivo effects of mutant RHOA on tumor formation in an orthotopic inoculation model.

Ras homolog family member A (RHOA) mutations are driver genes in diffuse-type gastric cancers (DGCs), and we previously revealed that RHOA mutations contribute to cancer cell survival and cell migration through their dominant negative effect on Rho-associated kinase (ROCK) signaling in vitro. However, how RHOA mutations contribute to DGC development in vivo is poorly understood. In the present study, the contribution of RHOA mutations to tumor morphology was investigated using an orthotopic xenograft model using the gastric cancer cell line MKN74, in which wild-type (WT) or mutated (Y42C and Y42S) RHOA had been introduced. When we conducted RNA sequencing to distinguish between the genes expressed in human tumor tissues from those in mouse stroma, the expression profiles of the tumors were clearly divided into a Y42C/Y42S group and a mock/WT group. Through gene set enrichment analysis, it was revealed that inflammation- and hypoxia-related pathways were enriched in the mock/WT tumors; however, cell metabolism- and cell cycle-related pathways such as Myc, E2F, oxidative phosphorylation and G2M checkpoint were enriched in the Y42C/Y42S tumors. In addition, the gene set related to ROCK signaling inhibition was enriched in the RHOA-mutated group, which indicated that a series of events are related to ROCK inhibition induced by RHOA mutations. Histopathological analysis revealed that small tumor nests were more frequent in RHOA mutants than in the mock or WT group. In addition, increased blood vessel formation and infiltration of macrophages within the tumor mass were observed in the RHOA mutants. Furthermore, unlike mock/WT, the RHOA-mutated tumor cells had little antitumor host reaction in the invasive front, which is similar to the pattern of mucosal invasion in clinical RHOA-mutated DGC. These transcriptome and pathological analyses revealed that mutated RHOA functionally contributes to the acquisition of DGC features, which will accelerate our understanding of the contribution of RHOA mutations in DGC biology and the development of further therapeutic strategies.

Hyaluronan preconditioning of monocytes/macrophages affects their angiogenic behavior and regulation of TSG-6 expression in a tumor type-specific manner.

Hyaluronan is a glycosaminoglycan normally present in the extracellular matrix in most tissues. Hyaluronan is a crucial player in many processes associated with cancer, such as angiogenesis, invasion, and metastasis. However, little has been reported regarding the action of hyaluronan on monocytes/macrophages (Mo/MØ) in tumor angiogenesis and its consequences on tumor development. In the present study, we investigated the effects of hyaluronan of different sizes on human Mo/MØ angiogenic behavior in colorectal and breast carcinoma. Invitro, the treatment of Mo/MØ with lysates and conditioned media from a breast but not from colorectal carcinoma cell line plus high-molecular weight hyaluronan induced: (a) an increased expression of angiogenic factors VEGF, IL-8, FGF-2, and MMP-2, (b) an increased endothelial cell migration, and (c) a differential expression of hyaluronan-binding protein TSG-6. Similar results were observed in Mo/MØ derived from breast cancer patients treated with tumor lysates. Besides, macrophages primed with high-molecular weight hyaluronan and inoculated in human breast cancer xenograft tumor increased blood vessel formation and diminished TSG-6 levels. In contrast, the effects triggered by high-molecular weight hyaluronan on Mo/MØ in breast cancer context were not observed in the context of colorectal carcinoma. Taken together, these results indicate that the effect of high-molecular weight hyaluronan as an inductor of the angiogenic behavior of macrophages in breast tumor context is in part consequence of the presence of TSG-6.

Biomaterial Platform To Establish a Hypoxic Metastatic Niche in Vivo.

Hypoxia is a hallmark of tumor microenvironments, exerting wide-ranging impacts on key processes of tumor progression and metastasis. However, our understanding of how hypoxia regulates these processes has been based primarily on studying the effects of hypoxia within the primary tumor. Recently, an increasing number of studies have suggested the importance of hypoxic regulation within metastatic target organs, but hypoxic metastatic niches in the body are difficult to access with current imaging techniques, hampering detailed in vivo investigation of hypoxia at metastatic sites. Here, we report an engineered biomaterial scaffold that is able to establish an in vivo hypoxic metastatic niche in a readily accessible area, enabling the investigation of hypoxic regulation at a metastatic site. We engineered hypoxic environments within microporous poly(lactide-co-glycolide) (PLG) scaffolds, which have previously been shown to act as premetastatic niche mimics, via the addition of CoCl2, a hypoxia-mimetic agent. When implanted into the subcutaneous region of mice, CoCl2-containing PLG (Co-PLG) scaffolds established hypoxic microenvironments, as evidenced by the stabilization of hypoxia-inducible factor 1α (HIF1α) and increased blood vessel formation in vitro and in vivo. Furthermore, implanted Co-PLG scaffolds were able to recruit 4T1 metastatic breast cancer cells. These results demonstrate that Co-PLG scaffolds can establish an in vivo hypoxic metastatic niche, providing a novel platform to investigate hypoxic regulation of disseminated tumor cells (DTCs) at target organs.

Fructose 1,6-Bisphosphate as a Protective Agent for Experimental Fat Grafting.

Fat grafting procedures are considered to be a promising regenerative, cell‐directed therapy; however, their survival is mainly influenced by ischemia condition. Fructose 1,6‐bisphosphate (FBP), as an intermediate in energy metabolism, has the potential to rescue cells and tissues from hypoxic‐ischemic circumstances. In the present study, human lipoaspirates were grafted subcutaneously into nude mice followed by a daily intraperitoneal injection of FBP at different doses for 7 days. Next, the grafts were harvested at different time points till 12 weeks postimplantation and were evaluated for cell viability and function, tissue revascularization and inflammatory cell infiltration using histological analysis, whole‐mount living tissue imaging, glycerol 3‐phosphate dehydrogenase activity assays, and quantitative analysis of gene expression. The results demonstrated that exogenous FBP administration could attenuate the volume and weight reduction of fat graft; meanwhile, FBP enhanced adipocyte viability and function, increased blood vessel formation, and decreased inflammation. Moreover, in vitro cell experiments showed that FBP could promote adipose‐derived stem cell viability and vascular endothelial growth factor (VEGF) mRNA expression in ischemia conditions. Our study indicates that FBP can be used as a protective agent for fat grafting and may be applied in stem cell‐based regenerative medicine. stem cells translational medicine 2019;8:606–616

Injectable Nanoclay Gels for Angiogenesis

The retention and sustained activity of therapeutic proteins at delivery sites are goals of regenerative medicine. Vascular endothelial growth factor (VEGF) has significant potential in promoting the growth and regeneration of blood vessels but is intrinsically labile. This is exacerbated by the inflammatory microenvironments at sites requiring regeneration. For VEGF to be efficacious it may require a carrier that stabilises it, protects it from degradation and retains it at a site of interest. In this study we tested the hypothesis that injectable nanoclay gels composed of Laponite XLGTM can stabilise VEGF and retain it in active form for therapeutic delivery. To achieve this, VEGF was incorporated in Laponite gels and its activity tested at a range of concentrations using in vitro cell culture tubulogenesis assays and in vivo angiogenesis assays. We found that VEGF-Laponite gels enhanced tubulogenesis in a dose-dependent manner in vitro. When administered subcutaneously in vivo Laponite was retained at an injection site for up to a period of three weeks and promoted a 4-fold increase in blood vessel formation compared with alginate or vehicle controls as confirmed by CD31 staining. Notably, in contrast to alginate, Laponite gels did not release VEGF, indicating a strong interaction between the growth factor and the nanoclay, and suggesting that Laponite enhancement of VEGF efficacy is due to its retention at an implantation site over a prolonged period. Our approach provides a robust method for delivery of bioactive recombinant VEGF without the necessity for complex hydrogel or protein engineering.

Targeting expression of adenosine receptors during hypoxia induced angiogenesis – A study using zebrafish model

Hypoxia is known to be a major player during pathological angiogenesis and adenosine as a negative feedback signaling to maintain oxygen delivery in pathological ischemic condition. We mimicked hypoxic condition and studied angiogenesis by inducing adenosine receptors using forskolin, a plant compound and NECA analogue of adenosine using zebrafish model. Vascular endothelial growth factor (VEGF) is known to play a key role during pathological angiogenesis and regulated by the factors HIF1a under hypoxic condition and recently Notch is proposed to play a negative feedback loop mechanism along with VEGF signaling but the role of adenosine receptor during the process is not known. We evaluated the mRNA expression of adenosine receptors (A1, A2a.1, A2a.2, A2b), HIF1a, VEGF A, VEGF R2, NRP1a, NOTCH 1a and DLL4 and the phenotypic variations of zebrafish embryos when treated with DAPT, γ-secretase inhibitor of Notch in addition to treating the embryos with SU5416, a VEGF receptor inhibitor. Upregulation of adenosine receptors (A1, A2a.1, A2a.2, A2b), HIF1a, VEGF A, VEGF R2, NRP1a, NOTCH1a and DLL4 was observed embryos were when treated with forskolin and NECA could possibly mimic hypoxic condition. Hatching and heart rate also increased with NECA and forskolin. SU5416 showed decreases in blood vessel formation and decreased adenosine receptors, VEGF, VEGFR2, HIF1a and NRP1a expression and DAPT, exhibited decreases in blood vessels and decreased NRP1a, NOTCH1a, DLL4 expression. These embryos developed with poor vasculature, tail bending, abnormal phenotypes and developmental delay. Forskolin treated with inhibitors showed increased blood vessel formation, normal phenotype, development and adenosine receptors (A1, A2a.1, A2a.2, A2b), HIF1a, VEGF A, VEGF R2, NRP1a, NOTCH 1a and DLL4 gene expression suggesting that adenosine mediated Notch and VEGF could play an important role during development and angiogenesis. Targeting VEGF and Notch signaling with adenosine receptors inhibitors which might have a therapeutic significance during hypoxia and abnormal angiogenesis.

Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated via mTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.