Tubulogenesis Assays Research Articles

Tetramethylpyrazine promotes osteo-angiogenesis during bone fracture repair

BackgroundNonunion following a long bone fracture has gained a lot of attention due to the dreadful impact on the life quality of tremendous patients. Recent data have demonstrated the important involvement of angiogenesis in improving fracture healing. Tetramethylpyrazine (TMP) is an active component of Chinese herbal medicine with various biological activities including pro-angiogenesis property. However, the activity and mechanism of action of TMP in osteo-angiogenesis during bone fracture repair bone fracture healing remain unknown. In this study, TMP was tested for its specific activities in rat aortic endothelial cells (RAECs) and fractured rat model.MethodsThe effect of TMP on angiogenesis and migration in RAECs was detected by conducting matrigel tubulogenesis assay and transwell assay. Histopathological changes were observed in the rats from each group using H&E staining. The levels of inflammation and coagulation markers in rats were evaluated by ELISA. The expression of osteogenesis-related genes in rats was assessed by RT-qPCR and western blotting.ResultsTMP promoted angiogenesis processes and migratory ability in RAECs. TMP improved histopathological changes in fractured rat model. The concentration of inflammatory markers (IL-2, IL-6, IL-1beta) in the serum of fractured rats were suppressed by TMP treatment. TMP also had the potential to inhibit blood coagulation in rat tibia fracture model. In addition, the expression and protein levels of osteogenesis-related markers (ALP, Runx2, and OPN-1) were elevated by TMP in the tissues from the fractured rats. In mechanism, TMP significantly promoted the activation of VEGF/FLK1 pathway in vitro and in vivo.ConclusionTMP accelerated the repair of bone fracture by promoting angiogenesis and osteogenesis.

NOS3 regulates angiogenic potential of human induced pluripotent stem cell-derived endothelial cells

Incorporation of blood capillaries in engineered tissues and their functional connection to host blood vessels is essential for success in engineering vascularized tissues, a process which involves spatial patterning of endothelial cells (ECs) to form functional and integrated vascular networks. Different types of ECs have been employed for vascular network formation and each source offers advantages and disadvantages. While ECs derived from induced pluripotent stem cells (iPSC-ECs) offer advantages over primary ECs in that they can be generated in large quantities for autologous applications, their suitability for disease modelling and cell replacement therapies is not well-explored. The present study compares the angiogenic capacity of iPSC-ECs and primary ECs (cardiac microvascular ECs and lymphatic microvascular ECs) using an in vitro tubulogenesis assay, revealing comparable performance in forming a pseudo-capillary network on Matrigel. Analysis of genes encoding angiogenic factors (VEGFA, VEGFC, VEGFD and ANG), endothelial cell markers (PECAM1, PCDH12 and NOS3) and proliferation markers (AURKB and MKI67) indicates a significant positive correlation between NOS3 mRNA expression levels and various tubulogenic parameters. Further experimentation using a CRISPR activation system demonstrates a positive impact of NOS3 on tubulogenic activity of ECs, suggesting that iPSC-ECs can be enhanced with endogenous NOS3 activation. Collectively, these findings highlight the potential of iPSC-ECs in generating vascularized tissues and advancing therapeutic vascularization.

Inhibition of microRNA-181c-5p rescues diabetes-impaired angiogenesis through activation of key mediators of angiogenesis, cell survival and novel genes, elmo3 and trib1

Abstract Introduction Patients with diabetes have impaired angiogenesis and poor coronary collateral vessel formation post-myocardial infarction (MI), which associates with higher mortality. There is a significant unmet clinical need for new agents that stimulate angiogenesis in response to ischaemia in diabetes. We have identified that miR-181c-5p has anti-angiogenic properties, but it’s role in diabetes remains unknown. Aim To elucidate the role of miR-181c-5p in diabetes-impaired angiogenesis. Methods Human coronary artery endothelial cells were transfected with a miR-181c-5p inhibitor (antimiR-181c-5p) or negative control (antimiR-Neg), exposed to glucose (5–25mM, 48h) then underwent Matrigel tubulogenesis assay or Boyden Chamber migration assay. Levels of proteins important for angiogenesis (e.g., VEGFA, p-ERK2, p-eNOS) were determined by Western Blot. Whole transcriptome sequencing was performed in vitro to identify novel gene targets of miR-181c-5p. In vivo, diabetic mice underwent hindlimb ischaemia or wound healing surgery and were injected with antimiR-181c-5p or antimiR-Neg. Tissues were extracted early (day 3) and late (days 10-14) post-surgery. Hindlimb blood-flow reperfusion was measured by Laser Doppler imaging. Hindlimb apoptosis was assessed by TUNEL and necrotic area was assessed by H&E. Wound area was calculated daily. Neovascularisation was assessed by CD31 (capillaries) and α-actin (arterioles) immunostaining. Results Inhibition of miR-181c-5p increased endothelial tubule number (+28%, P<0.01), tubule length (+12%, P<0.01) and cell migration (+67%, P<0.05). This associated with increased VEGFA (+21%, P<0.05) and p-ERK2 (+32%, P<0.05). Whole transcriptome and pathway analysis revealed changes to angiogenesis pathways and identified a first-time involvement of genes Elmo3 and Trib1 in the pro-angiogenic action of antimiR-181c-5p in diabetes. In vivo, inhibition of miR-181c-5p increased blood flow reperfusion to ischaemic hindlimbs (+30%, P<0.001) and arteriolar density (+45%, P<0.05) in diabetic mice. Mechanistically, this was associated with early changes to mediators of angiogenesis, Erk2 mRNA (+35%, P<0.05), p-ERK2 (+35%, P<0.05) and Trib1 mRNA (+80%, P<0.05); cell survival, Bcl-2 mRNA (+44%, P<0.05); and late apoptotic clearance, Elmo3 mRNA (+57%, P<0.001). Furthermore, this was also associated with an increase in late stage hindlimb apoptosis (+94%, P<0.05) and reduced necrotic area (-90%, P<0.05) in diabetic mice. Inhibition of miR-181c-5p increased diabetic wound closure (+22%, P<0.01), wound capillaries (+61%, P<0.05), Bcl-2 mRNA (+52%, P<0.05) and Elmo3 mRNA (+50%, P<0.05) in diabetic wounds. Conclusions Inhibition of miR-181c-5p rescues diabetes-impaired angiogenesis by activation of angiogenesis and cell survival mediators, and through novel genes, Trib1 and Elmo3. Our findings have implications for a novel miRNA-based strategy that improves myocardial neovascularisation and the prognosis of diabetic patients post-MI.

Ex Vivo Evaluation of Antiangiogenic Drugs in Oral Cancer: Potential Implications for Targeting Vasculogenic Mimicry.

Oral squamous cell carcinoma (OSCC) is characterized by early metastasis, clinical resistance and poor prognosis. Recently, we showed that aggressive OSCC cells co-express endothelial cell markers and can form tube-like structures, known as vasculogenic mimicry (VM), a process associated with poor prognosis in head and neck cancers. Given the limited success of current antiangiogenic therapy in treating OSCC, this study sought to explore the efficiency of these drugs in targeting an ex vivo model of VM. OSCC cell lines from the tongue and floor of the mouth in addition to human endothelial cells were used. The treatments comprised a set of clinically relevant antiangiogenic drugs: sorafenib, sunitinib, and axitinib, which were administered in different doses. Multiple ex vivo approaches including cell tubulogenesis, proliferation, apoptosis, and migration assays were used. Although these drugs inhibited the formation of endothelial cell capillaries, they showed clear differential effects on OSCC cell-derived VM and cell morphology. Sorafenib inhibited the tubulogenesis of aggressive OSCC cells compared with the limited effect of sunitinib and axitinib. Furthermore, our data consistently demonstrated a preferential efficacy of certain drugs over others. Sorafenib and sunitinib exhibited anti-cancer effects on tumor cell proliferation, apoptosis, and cell migration, compared with the limited effect of axitinib. The antiangiogenic drugs, except sorafenib, had limited effect on VM formation in vitro and exhibited varying anti-cancer effects on OSCC cells. These data support the notion that VM formation may in part explain the development of drug resistance in OSCC cells.

Extracellular vesicles from Aggregatibacter actinomycetemcomitans exhibit potential antitumorigenic effects in oral cancer: a comparative in vitro study

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

Four sides to the story: A proteomic comparison of liquid-phase and matrix-bound extracellular vesicles in 2D and 3D cell cultures.

Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.

EEF1A2 promotes HIF1A mediated breast cancer angiogenesis in normoxia and participates in a positive feedback loop with HIF1A in hypoxia.

The eukaryotic elongation factor, EEF1A2, has been identified as an oncogene in various solid tumors. Here, we have identified a novel function of EEF1A2 in angiogenesis. Chick chorioallantoic membrane, tubulogenesis, aortic ring, Matrigel plug, and skin wound healing assays established EEF1A2's role in angiogenesis. Higher EEF1A2 levels in breast cancer cells enhanced cell growth, movement, blood vessel function, and tubule formation in HUVECs, as confirmed by ex-ovo and in-vivo tests. The overexpression of EEF1A2 could be counteracted by Plitidepsin. Under normoxic conditions, EEF1A2 triggered HIF1A expression via ERK-Myc and mTOR signaling in TNBC and ER/PR positive cells. Hypoxia induced the expression of EEF1A2, leading to a positive feedback loop between EEF1A2 and HIF1A. Luciferase assay and EMSA confirmed HIF1A binding on the EEF1A2 promoter, which induced its transcription. RT-PCR and polysome profiling validated that EEF1A2 affected VEGF transcription and translation positively. This led to increased VEGF release from breast cancer cells, activating ERK and PI3K-AKT signaling in endothelial cells. Breast cancer tissues with elevated EEF1A2 showed higher microvessel density. EEF1A2 exhibits angiogenic potential in both normoxic and hypoxic conditions, underscoring its dual role in promoting EMT and angiogenesis, rendering it a promising target for cancer therapy.

Microporous Hydroxyapatite-Based Ceramics Alter the Physiology of Endothelial Cells through Physical and Chemical Cues.

Incorporation of silicate ions in calcium phosphate ceramics (CPC) and modification of their multiscale architecture are two strategies for improving the vascularization of scaffolds for bone regenerative medicine. The response of endothelial cells, actors for vascularization, to the chemical and physical cues of biomaterial surfaces is little documented, although essential. We aimed to characterize in vitro the response of an endothelial cell line, C166, cultivated on the surface CPCs varying either in terms of their chemistry (pure versus silicon-doped HA) or their microstructure (dense versus microporous). Adhesion, metabolic activity, and proliferation were significantly altered on microporous ceramics, but the secretion of the pro-angiogenic VEGF-A increased from 262 to 386 pg/mL on porous compared to dense silicon-doped HA ceramics after 168 h. A tubulogenesis assay was set up directly on the ceramics. Two configurations were designed for discriminating the influence of the chemistry from that of the surface physical properties. The formation of tubule-like structures was qualitatively more frequent on dense ceramics. Microporous ceramics induced calcium depletion in the culture medium (from 2 down to 0.5 mmol/L), which is deleterious for C166. Importantly, this effect might be associated with the in vitro static cell culture. No influence of silicon doping of HA on C166 behavior was detected.

Trajectory of hiPSCs derived neural progenitor cells differentiation into dermal papilla-like cells and their characteristics

Dermal papilla cells (DPCs) play roles in key functions of the epidermis such as hair generation. The use of human induced pluripotent cells (hiPSCs) makes it possible to obtain DP-like cells and study the molecular mechanisms of DPC development during embryogenesis. In this work, we studied the phenotypic trajectory of hiPSCs during their differentiation into DP-like cells and evaluated the epithelial-mesenchymal interaction potential of the resulting cell line. Specifically, we differentiated hiPSCs into neural progenitor cells (NPCs) and subsequently into DP-like cells. Analysis of bulk RNA-seq data during this process enabled us to observe gene expression dynamics during five stages of dermal differentiation. Furthermore, functional assays (organoids in both collagen gels and hanging drop cultures and tubulogenesis assays) revealed that the dermal cell lines we generated could interact with epidermal cells.

Huzhangqingmaiyin protected vascular endothelial cells against cerebral small vessel disease through inhibiting inflammation

Ethnopharmacological relevanceHuzhangqingmaiyin (HZQMY) is a Chinese medicine formula used to treat small vessel disease, but the mechanism is unclear. Aim of the studyThis study aimed to reveal the protective effects of HZQMY on human brain microvascular endothelial cells (HBMECs) and explore the potential targets and mechanistic pathways using network pharmacology on treating cerebral small vessel disease (CSVD). Materials and methodsHBMECs were cultured in vitro and an endothelial cell injury model was constructed by hypoxia for 12 h followed by reoxygenation for 8 h (H/R). Cell viability was measured by CCK-8 assay, migration ability of cells was detected by scratch assay, angiogenesis ability of endothelial cells was detected by tubulogenesis assay. Meanwhile, JC-1 staining was employed to determine the alteration of mitochondrial membrane potential, and finally, cell apoptosis was assessed by flow cytometry. To further explore the mechanism of action of HZQMY, the target proteins of a candidate active compound was first collected from the traditional Chinese medicine systems pharmacology database with analytical platform and Swiss target prediction database (www.swisstargetprediction.ch) by HPLC/MS determination of its main active components. CSVD associated targets were retrieved from four disease associated targets databases, OMIM, DisGenNET, GeneCards and GeneCLip, respectively. Using the website String, the genes overlapped between HZQMY and CSVD were imported into the database, PPI network plots were drawn using Cytoscape software. GO and KEGG analyses were performed to explore the possible pathways and targets of HZQMY. Its most probable targets were further explored with molecular docking and verified. ResultsHZQMY at 0.5–2 μg/mL concentration range could promote cell proliferation, cell migration, angiogenesis, reduce mitochondrial membrane potential damage as well as inhibit apoptosis. Besides that, 29 active compounds were detected from HZQMY, including key components such as quercetin, polydatin, kaempferol, isorhamnetin and resveratrol. Core targets that might include IL-1β、ICAM-1、VCAM-1 and VEGF and so on. ConclusionsHZQMY could regulate the levels of key targets such as IL-1β、ICAM-1、VCAM-1 and VEGF, so as to achieve the purpose of treating CSVD.

Evaluation of Endothelial Progenitor Cell Characteristics as Clinical Biomarkers for Elderly Patients with Ischaemic Stroke.

Ageing impairs endothelial function and predisposes the person to ischaemic stroke (IS). Endothelial progenitor cells (EPCs) repair endothelial damage and induce post-ischaemic neovascularisation. Given the prevalence of IS in older population, this study explored whether changes in EPC number and function may reliably predict the type or outcome of stroke in patients ≥ 65years of age. For this, blood samples were collected once from healthy volunteers (HVs, n = 40) and four times (admission and days 7, 30 and 90 post-stroke) from participants with lacunar (n = 38) or cortical (n = 43) stroke. EPCs were counted with flow cytometry and defined as non-haematopoietic cells (CD45-) expressing markers for stemness (CD34 +), immaturity (CD133 +) and endothelial maturity (KDR +). Clonogenesis, tubulogenesis, migration and proliferation assays were performed as measures of EPC functionality. Biochemical profile of plasma inflammatory and angiogenic agents were studied using specific ELISAs. Primary outcome was disability or dependence on day 90 post-stroke, assessed by the modified Rankin Scale (mRS). Compared to HVs, EPC numbers were higher in stroke patients at all time points studied, reaching significance at baseline and day 30. No differences in EPC counts and functionality were observed between lacunar and cortical stroke groups at any time. Plasma endostatin, PDGF-BB, TNF-α and VEGF levels were higher in stroke patients vs HVs. Patient outcome, evaluated by mRS on day 90 post-stroke, did not correlate with EPC count or functionality. Baseline EPC counts may serve as a diagnostic marker for stroke but fail to distinguish between different stroke subtypes and predict post-stroke outcome.

Taohong Siwu Decoction Promotes Osteo-Angiogenesis in Fractures by Regulating the HIF-1α Signaling Pathway.

Background Vascular damage is a major consequence of bone fracture. Taohong Siwu decoction (TSD) can raise the expression of vascular endothelial growth factor (VEGF) in fracture healing. However, its molecular mechanism in promoting angiogenesis is still unknown. The aim of this study was to investigate the potential mechanisms of TSD in the regulation of osteo-angiogenesis in fracture healing. Methods A rat tibial fracture model was established. After low- (4.5 g·kg−1), medium- (9 g·kg−1), and high-dose TSD (18 g·kg−1) and panax notoginsenoside (25 mg kg−1) treatment, hematoxylin-eosin staining was employed to visualize pathological changes in bone tissues. The levels of cytokines (interleukin (IL)-2, tumor necrosis factor-α (TNF-α), IL-6, and IL-1β), thromboxane B2 (TXB2), and 6 ketone prostaglandin F1α (6-Keto-PGF1α) were quantified by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to identify the rat aortic endothelial cells (RAECs). Control serum, 10% TSD-containing serum, and 10% TSD-containing serum combined with hypoxia-inducible factor-1α (HIF-1α) inhibitor were used to treat the RAECs and rat osteoblasts. Transwell migration assay was utilized to examine the migration of the RAECs. The Matrigel tubulogenesis assay was used for the assessment of angiogenesis. The expression of angiogenesis- (von Hippel-Lindau tumor suppressor (VHL), HIF-1α, VEGF, angiopoietin-2 (Ang-2), and pVHL) and osteogenesis-related (alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin-1 (OPN-1)) protein and gene was detected by western blot and quantitative real-time PCR (qRT-PCR). Results Compared with the model group, TSD increased the trabecular bone areas, numbers, and thicknesses in fractured rats. In the plasma, the levels of cytokines and TXB2 in the middle- and high-dose TSD group were significantly lower than those in the model group (P < 0.01). The 6-keto-PGF1α content was increased by middle- and high-dose TSD intervention (P < 0.01). Compared to the control serum group, the angiogenesis and migration of the RAECs were enhanced in the TSD group (P < 0.001). The expression of HIF-1α, VEGF, and Ang-2 in the TSD group upregulated significantly (P < 0.001). VHL and pVHL were inhibited under TSD-containing serum treatment (P < 0.001). ALP, Runx2, and OPN-1 were increased obviously in the TSD group (P < 0.001). Nevertheless, the HIF-1α inhibitor reversed these changes (P < 0.001). Conclusion TSD promotes angiogenesis and osteogenesis by regulating the HIF-1α signaling pathway. Meanwhile, it can effectively reduce the risk of inflammation and improve blood circulation.

TNFAIP3 mediates FGFR1 activation-induced breast cancer angiogenesis by promoting VEGFA expression and secretion.

To investigate the role and mechanism of TNF-inducible protein 3(TNFAIP3) in breast cancer angiogenesis induced by fibroblast growth factor receptor1 (FGFR1) activation. The immunohistochemical assay was used to detect the expression of vascular endothelial cell marker CD31 and CD105 in mice DCIS.COM-iFGFR1 transplanted tumor (previously established by our group). The effects of TNFAIP3 knockout/knockdown breast cancer cell lines on angiogenesis, migration, and invasion of Human Umbilical Vein Endothelial Cells (HUVEC) were detected by the tubulogenesis and Trewells assay. RNA-seq analysis of TNFAIP3 downstreams differential genes after TNFAIP3 knockdown. The expression and secretion of VEGFA after FGFR1 activation in breast cancer cells were detected by qPCR, Western blot, and ELISA. Immunohistochemistry showed that TNFAIP3 knockout inhibited the expression of CD31 and CD105 in DCIS grafted tumors promoted by FGFR1 activation. Tubulogenesis and Trewells experiments showed that TNFAIP3 gene knockout/knockdown inhibited the angiogenesis, migration, and invasion of HUVEC cells promoted by FGFR1 activation. qPCR assay showed that VEGFA mRNA level in the TNFAIP3 knockdown cell line was significantly down-regulated (p < 0.05). qPCR, Western blot and ELISA results showed that TNFAIP3 gene knockout/knockdown could inhibit the expression and secretion of VEGFA in breast cancer cells induced by FGFR1 activation. TNFAIP3 promotes breast cancer angiogenesis induced by FGFR1 activation through the expression and secretion of VEGFA.

Proinflammatory mediators, TNFα, IFNγ, and thrombin, directly induce lymphatic capillary tube regression.

In this work, we sought to investigate the direct effects of proinflammatory mediators on lymphatic endothelial cell (LEC) capillaries and whether they might induce regression. Our laboratory has developed novel in-vitro, serum-free, lymphatic tubulogenesis assay models whereby human LEC tube networks readily form in either three-dimensional collagen or fibrin matrices. These systems were initially conceptualized in the hopes of better understanding the influence of proinflammatory mediators on LEC capillaries. In this work, we have screened and identified proinflammatory mediators that cause regression of LEC tube networks, the most potent of which is TNFα (tumor necrosis factor alpha), followed by IFNγ (interferon gamma) and thrombin. When these mediators were combined, even greater and more rapid lymphatic capillary regression occurred. Surprisingly, IL-1β (interleukin-1 beta), one of the most potent and pathologic cytokines known, had no regressive effect on these tube networks. Finally, we identified new pharmacological drug combinations capable of rescuing LEC capillaries from regression in response to the potent combination of TNFα, IFNγ, and thrombin. We speculate that protecting lymphatic capillaries from regression may be an important step toward mitigating a wide variety of acute and chronic disease states, as lymphatics are believed to clear both proinflammatory cells and mediators from inflamed and damaged tissue beds. Overall, these studies identify key proinflammatory mediators, including TNFα, IFNγ, and thrombin, that induce regression of LEC tube networks, as well as identify potential therapeutic agents to diminish LEC capillary regression responses.

3D Printed Scaffolds Incorporated with Platelet‐Rich Plasma Show Enhanced Angiogenic Potential while not Inducing Fibrosis

AbstractSuccessful therapeutic strategies for wound healing rely on proper vascularization while inhibiting fibrosis. However, scaffolds designed for skin tissue engineering generally lack the biochemical cues that can enhance their vascularization without inducing fibrosis. Therefore, the objective of this work is to incorporate platelet‐rich plasma (PRP), a natural source of angiogenic growth factors, into a gelatin methacrylate (GelMA) hydrogel, yielding a bioink that can subsequently be used to 3D print a novel regenerative scaffold with defined architecture for skin wound healing. A PRP‐activated bioink is successfully 3D printed, and the resulting scaffolds present similar structural, rheological, and mechanical properties compared to GelMA‐only scaffolds. Furthermore, 3D printed PRP‐activated scaffolds facilitate controlled release of PRP‐derived growth factors for up to 14 days, presenting superior angiogenic potential in vitro (e.g., tubulogenesis assay) and in vivo (chick chorioallantoic membrane) compared to GelMA‐only scaffolds, while not inducing a myofibroblastic phenotype in fibroblasts (e.g., α‐smooth muscle actin expression). This disruptive technology offers the opportunity for a patient's autologous growth factors to be incorporated into a tailored 3D‐printed scaffold in theatre prior to implantation, as part of a single‐stage procedure, and has potential in other tissue engineering applications in which enhanced vascularization with limited fibrosis is desired.

Onion Peel Extract Inhibits Cancer Cell Growth and Progression through the Roles of L1CAM, NF-κB, and Angiogenesis in HT-29 Colorectal Cancer Cells.

Colorectal cancer (CRC) is an aggressive malignancy. Critical mechanisms that support CRC progression include cell migration, invasion, metastasis, and angiogenesis, which is associated with L1 cell adhesion molecule (L1CAM) and nuclear factor-kappa B (NF-κB) signaling pathways. In this study, viability of HT-29 cells and human umbilical vein endothelial cells (HUVECs) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and cell apoptosis was investigated by flow cytometry assays. HT-29 cell migration and invasion were observed by wound healing and Transwell invasion assays, respectively, and tube formation of HUVECs was observed by tubulogenesis assays. L1CAM and NF-κB protein expressions in HT-29 cells treated with onion peel extract were determined by indirect immunofluorescence. Results showed that high dose treatments of onion peel extract inhibited cell viability of both HT-29 cells and HUVECs, induced HT-29 cell apoptosis, and inhibited HT-29 cell migration and invasion. Moreover, onion peel extract decreased total HUVEC tube length and, at a concentration of 10 μg/mL, showed potential to downregulate L1CAM and NF-κB. In conclusion, onion peel extract inhibits HT-29 cell growth, migration, and invasion through suppressing pathways related to angiogenesis downstream of L1CAM-activated NF-κB.

Static and dynamic culture of human endothelial cells encapsulated inside alginate-gelatin microspheres

This study aimed to explore the angiogenesis potential of human endothelial cells encapsulated inside alginate-gelatin microspheres under static and dynamic culture systems after 7 days.Human umbilical vein endothelial cells were encapsulated inside alginate (1%) and gelatin (1.2%) using an electrostatic encapsulation method. Cells were incubated for 7 days in vitro. The cell survival rate was measured using the MTT assay. The expression of VEGFR-2 and von Willebrand factor genes was studied by real-time PCR assay. Using western blot analysis, we monitored the protein contents of VEGFR-2, vWF, and Caspase 3. The levels of SOD and GPx enzymes were calculated using biochemical kits. Angiogenesis potential was assessed using in vitro Matrigel assay.Data showed an increased survival rate in encapsulated cells cultured under the static condition compared to the conventional 2D condition (p < 0.05). The culture of encapsulated cells under a dynamic bioreactor system did not alter cell viability. Compared to the dynamic culture system, the incubation of encapsulated cells in the static culture system swelled the microspheres (p < 0.05). Both dynamic and static culture models increased the expression of VEGFR-2 and von Willebrand factor in encapsulated cells compared to 2D culture (p < 0.05), showing enhanced functional maturation. Data showed a significant increase of vWF and reduction of apoptosis marker Caspase in the dynamic culture system (p < 0.05). The levels of SOD and GPx were significantly increased in dynamic and static culture models as compared to the control 2D group (p < 0.05). In vitro tubulogenesis assay showed significant induction of angiogenesis in dynamic encapsulated HUVECs indicated with a large number of vascular tubes and arborized ECs compared to the control and static encapsulated HUVECs (p < 0.05).The current study suggests a bioreactor dynamic system is a reliable approach, similar to a static condition, for the expansion of encapsulated human ECs in a 3D milieu.

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with “hepatoblast-like” appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.

Juxtaposition of Mesenchymal Stem Cells with Endothelial Progenitor Cells Promoted Angiogenic Potential Inside Alginate-Gelatin Microspheres.

Purpose: Here, we investigated the angiogenic potential of endothelial progenitor cells juxtaposed with mesenchymal stem cells (MSCs) inside alginate-gelatin microspheres with stromal derived factor-1α (SDF-1 α) for 7 days. Methods: Six encapsulated groups were allocated including endothelial progenitor cells (EPCs), EPCs/SDF-1α, MSCs, MSCs/SDF-1α, EPCs+MSCs and EPCs+MSCs/SDF-1α. Cells were encapsulated with a mixture of 1% alginate and 2% gelatin hydrogel. Cell survival was examined by MTT assay. Endothelial differentiation was determined by flow cytometry and ELISA. Tubulogenesis assay and Ac-Dil-LDL uptake were used to detect functional activity. Cell migration was analyzed by Transwell insert and gelatin zymography analyses. By using real-time polymerase chain reaction (PCR), we measured the transcription of Akt and PK1. Results: We found an increase in cell viability in MSCs/SDF-1α microspheres compared to EPCs group (P <0.05). EPC/MSCs co-culture contributed to the increase of CD133+ cells while we found high CD31 levels in MSCs group (P <0.05). Juxtaposition of EPC with MSCs increased tubulogenesis compared to SDF-1a-free condition (P <0.001). SDF-1α had the potential to increase in AC-LDL uptake in MSCs and EPCs/MSCs groups. Cells migration and MMP-9 activities increased after treatment with SDF-1α. SDF-1α upregulated PK1 and Akt in encapsulated cells, especially in a co-culture system. Conclusion: Alginate-gelatin microspheres could alter the angiogenic potential of progenitor cells in the presence of SDF-1α

Moderately Acidic pH Promotes Angiogenesis: An In Vitro and In Vivo Study

IntroductionThis study evaluated the effect of different pH values of 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4 on angiogenesis. MethodsEndothelial cells were isolated from the mice molar teeth and placed in 42 Matrigel (Corning, NY)-coated wells, which were prepared and divided into 6 groups (n = 7). Synthetic tissue fluid was prepared and divided into 6 parts, and their pH values were adjusted to 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4. A 2-mL volume from each group was diluted in the growth medium at a ratio of 1:3 and used for tubulogenesis assay. Forty-two 6-week-old mice in 6 groups (n = 7) were used for choroidal neovascularization (CNV). A 2-μL volume from each group or saline (control) was delivered by intravitreal injection on the day of laser application and 1 week later. Data on the number of nodes, the total length of the branches, and CNV areas (μm2) were determined using ImageJ software (National Institutes of Health, Bethesda, MD) and analyzed with 1-way analysis of variance and post hoc Tukey tests. The correlation was assessed between the tested variables. ResultsThe number of nodes decreased with changes in pH values as follows: 6.4 > 5.4 > 7.4 > 8.4 > 9.4 > 4.4. The total branch length decreased with pH value changes as follows: 6.4 > 4.4 > 6.4 > 7.4 > 8.4 > 9.4, and the CNV areas decreased with pH value changes as follows: 6.4 > 5.4 > 4.4 > 7.4 > 8.4 > 9.4. ConclusionsModerately acidic pH values (5.4 and 6.4) enhanced angiogenesis, whereas moderately alkaline pH values (8.4 and 9.4) suppressed angiogenesis.