Formation Of Capillary-like Structures Research Articles

A novel 3D cardiac microtissue model for investigation of cardiovascular complications in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease that affects not only the joints but also has significant cardiovascular (CV) manifestations. The mechanistic interplay between RA and cardiovascular complications is not yet well understood due to the lack of relevant in vitro models. In this study, we established RA cardiac microtisses (cMTs) from iPSC-derived cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs) to investigate whether this fully human 3D multicellular system could serve as a platform to elucidate the connection between RA and CV disorders.MethodsPBMC and FLS from healthy and RA donors were reprogrammed to hiPSCs with Sendai vectors. hiPSCs pluripotency was assessed by IF, FACS, spontaneous embryoid bodies formation and teratoma assay. hiPSCs were differentiated to cardiac derivatives such as CMs, ECs and CFs, followed by cell markers characterizations (IF, FACS, qRT-PCR) and functional assessments. 3D cMTs were generated by aggregation of 70% CMs, 15% ECs and 15% CFs. After 21 days in culture, structural and metabolic properties of 3D cMTs were examined by IF, qRT-PCR and Seahorse bioanalyzer.ResultshiPSCs demonstrated typical colony-like morphology, normal karyotype, presence of pluripotency markers, and ability to differentiate into cells originating from all three germ layers. hiPSC-CMs showed spontaneous beating and expression of cardiac markers (cTnT, MYL7, NKX2.5, MYH7). hiPSC-ECs formed sprouting spheres and tubes and expressed CD31 and CD144. hiPSC-CFs presented spindle-shaped morphology and expression of vimentin, collagen 1 and DDR2. Self-aggregation of CMs/ECs/CFs allowed development of contracting 3D cMTs, demonstrating spherical organization of the cells, which partially resembled the cardiac muscle, both in structure and function. IF analysis confirmed the expression of cTnT, CD31, CD144 and DDR2 in generated 3D cMTs. RA cMTs exhibited significantly greater formation of capillary-like structures, mimicking enhanced vascularization—key RA feature—compared to control cMTs. Seahorse examination of cMTs revealed changes in mitochondrial and glycolytic rates in the presence of metabolic substrates and inhibitors.ConclusionsThe cMTs model may represent an advanced human stem cell-based platform for modeling CV complications in RA. The highly developed capillary-like structures observed within RA cMTs highlight a critical feature of inflammation-induced CV dysfunction in chronic inflammatory diseases.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Angiogenesis and full thickness wound repair in a cell sheet-based vascularized skin substitute

Skin tissue engineering is undergoing tremendous expansion as a result from clinical needs, mandatory replacement of animal models and development of new technologies. Many approaches have been used to produce vascularized skin substitutes for grafting purposes showing the presence of capillary-like structures but with limited analysis of their in vitro maturation and plasticity. Such knowledge is however important for the development of tissue substitutes with improved implantation success as well as for validation of vascularization in vitro models, including as a readout in pharmacological analyses. For optimal interactions of cells with microenvironment and vasculature, we here used a cell sheet approach consisting in the sole production of matrix by the cells. In this context, we limited the density of endothelial cells seeded for self-assembly and rather relied on the stimulation of angiogenesis for the development of an extensive connected microvascular-like network. After detailed characterization of this network, we challenged its plasticity both during and after establishment of the skin substitute. We show that fine tuning of VEGF concentration and time of application differentially affects formation of capillary-like structures and their perivascular coverage. Furthermore, we performed a deep wound assay that displayed tissue repair and angiogenesis with unique characteristics of the physiological process. These studies demonstrate the importance of cell-derived microenvironment for the establishment of mature yet dynamic vascularized skin models allowing a wide range of pharmacological and basic investigations. Statement of significanceThe significant advancements in organ-on-chips and tissue engineering call for more relevant models including microvascularization with remodeling potential. While vascularized skin substitutes have been developed for years, focus has primarily been on the impact of microvascularization on implantation rather than on its in vitro characterization. We here developed a cell sheet-based vascularized skin substitute relying on angiogenesis, i.e. growth of vessel-like structures within the 3D model, rather than solely on endothelial cell self-assembly. We then characterized :1/ vascularization after modulation of angiogenic factor VEGF during the substitute construction; -2/ angiogenesis associated to tissue repair after deep mechanical wounding. These studies establish a solid physiologically relevant model for further investigation of skin cell interactions and in vitro wound healing.

The effects of the activation of TLR2/TLR1 on in vitro angiogenesis in an immortalized ovine luteal endothelial cell line.

Activation of TLR2/TLR1 alters in vitro formation of capillary-like structures and induces inflammatory processes in ovine luteal endothelial (OLENDO) cells. Postpartum bacterial infections of the uterus affect uterine physiology and ovarian activity, causing fertility problems. The outer membrane component of Gram-negative bacteria, lipopolysaccharide, is involved in the initiation of the local inflammatory processes, and other bacterial toxins, particularly lipopeptides, have also been shown to be potent cytokine inducers, acting via Toll-like receptor-2 (TLR2). However, the possible adverse effects of TLR2 on ovarian and luteal activities have not yet been investigated in depth. The strong expression of TLR2 in the blood vessels of the corpus luteum led us to hypothesize that TLR2 activation might participate in the disruption of luteal vascular functionality. Therefore, we analyzed the effects of Pam3CSK4 (Pam3CysSerLys4), a synthetic triacylated lipopeptide and TLR2/TLR1 ligand, on the functionality of gap junctional intercellular communication (GJIC), endothelial cell invasion, and in vitro capillary-like network formation in an immortalized ovine luteal endothelial (OLENDO) cell line. Pam3CSK4 treatment of OLENDO cells disrupted in vitro tube formation but had no effect on GJIC or migration of OLENDO cells. Furthermore, Pam3CSK4 induced the expression of NFKB, IL6, and IL8 in OLENDO cells. Additionally, the basal availability of TLRs (TLR1-10) and TLR co-receptors (MYD88, LY96/MD2, and CD14) in OLENDO cells was confirmed by conventional PCR. Finally, the activation of TLR2/TLR1 appears to alter in vitro formation of capillary-like structures and induce inflammatory processes in OLENDO cells.

The role of Plaur-miR1-5p encoded within the urokinase receptor gene (Plaur) in angiogenesis

Angiogenesis plays a crucial role in tissue and organ regeneration by supplying essential nutrients and oxygen through the development of new blood vessels. Mesenchymal stem/stromal cells release extracellular vesicles that actively contribute to angiogenesis by carrying pro-angiogenic growth factors and microRNAs. MicroRNAs, small non-coding RNA molecules, are central players in angiogenesis, affecting endothelial cell proliferation, specialization, migration, apoptosis, and post-transcriptional gene expression.In the present study, we investigated the impact of extracellular vesicles containing Plaur-miR1- 5p microRNAs on angiogenesis, specifically focusing on its initial stages: vascular cell migration and the formation of capillary-like structures. Recently we discovered Plaur-miR1-5p, which is encoded within the urokinase receptor gene (Plaur). However, the functions of this microRNA remain largely unexplored. Using a vascular ring model embedded in Matrigel, we demonstrate that Plaur-miR1-5p is encapsulated within extracellular vesicles and plays a regulatory role in capillary-like structure formation. Moreover, applying bioinformatic analysis, we have identified potential target genes of Plaur-miR1-5p that participate in the regulation of angiogenesis.This study advances our comprehension of the fundamental processes governing angiogenesis, particularly the involvement of extracellular vesicles and microRNAs. Moreover, it sheds light on the functional aspects ofthe Plaur gene, contributing to a more profound understanding of its role in regulation of angiogenesis.

Effect of Heparan Sulfate on Vasculogenesis and Dentinogenesis of Dental Pulp Stem Cells

IntroductionHeparan sulfate (HS) is a major component of dental pulp tissue. We previously reported that inhibiting HS biosynthesis impedes endothelial differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanisms by which exogenous HS induces DPSC differentiation and pulp tissue regeneration remain unknown. This study explores the impact of exogenous HS on vasculogenesis and dentinogenesis of DPSCs both in vitro and in vivo. MethodsHuman-derived DPSCs were cultured in endothelial and odontogenic differentiation media and treated with HS. Endothelial differentiation of DPSCs was investigated by real-time polymerase chain reaction and capillary sprouting assay. Odontogenic differentiation was assessed through real-time polymerase chain reaction and detection of mineralized dentin-like deposition. Additionally, the influence of HS on pulp tissue was assessed with a direct pulp capping model, in which HS was delivered to exposed pulp tissue in rats. Gelatin sponges were loaded with either phosphate-buffered saline or 101–102 μg/mL HS and placed onto the pulp tissue. Following a 28-day period, tissues were investigated by histological analysis and micro–computed tomography imaging. ResultsHS treatment markedly increased expression levels of key endothelial and odontogenic genes, enhanced the formation of capillary-like structures, and promoted the deposition of mineralized matrices. Treatment of exposed pulp tissue with HS in the in vivo pulp capping study induced formation of capillaries and reparative dentin. ConclusionsExogenous HS effectively promoted vasculogenesis and dentinogenesis of DPSCs in vitro and induced reparative dentin formation in vivo, highlighting its therapeutic potential for pulp capping treatment.

Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.

Evaluation of the antitumoral effects of the mesoionic compound MI-D: Implications for endothelial cells viability and angiogenesis inhibition

Angiogenesis is considered one of the hallmarks of cancer, assisting tumor progression and metastasis. The mesoionic compound, MI-D, can induce cell death and provoke cytoskeletal and metabolic changes in cancer cells. Using in vitro and in vivo models, this study aimed to evaluate the effects of MI-D on the viability of human endothelial cells (EC) and its ability to inhibit tumor-induced angiogenesis induced by tumoral cells. For in vitro analysis, colon carcinoma (HT29) and endothelial (EA.hy926) cells were used as the tumoral and angiogenesis models, respectively. To evaluate cytotoxicity, methylene blue viability stain and annexin-V/7AAD tests were performed with both cell types. For the angiogenesis experiments, scratch wound healing and capillary tube-like formation assays were performed with the EC. The in vivo tests were performed with the chorioallantoic membrane (HET-CAM) methodology, wherein gelatin sponge implants containing MI-D (5, 25, and 50 μM), HT29 cells, or both were grafted in the CAM. Our data showed that MI-D induced apoptosis in both endothelial and colon carcinoma cells, with a strong cytotoxic effect on the tumoral lineage. The drug inhibited the EC's migration and capillary-like structure formation in vitro. In the HET-CAM assays, MI-D reduced the number of blood vessels in the membrane when grafted alone and accompanied by tumor cells. In this study, MI-D interfered in important steps of angiogenesis, such as maintenance of endothelial cell viability, migration, formation of capillary-like structures, as well tumor-induced neovascularization, reinforcing the hypothesis that MI-D might act as an inhibitor of angiogenesis, and a potential antitumor agent.

Diverse effects of prostacyclin on angiogenesis-related processes in the porcine endometrium

Angiogenesis is important for endometrial remodeling in mature females. The endometrium synthesizes high amounts of prostacyclin (PGI2) but the role of PGI2 in angiogenesis-related events in this tissue was not fully described. In the present study, porcine endometrial endothelial (pEETH) cells and/or a swine umbilical vein endothelial cell line (G1410 cells) were used to determine the regulation of PGI2 synthesis and PGI2 receptor (PTGIR) expression by cytokines and to evaluate the effect of PGI2 on pro-angiogenic gene expression, intracellular signaling activation, cell proliferation and migration, cell cycle distribution, and capillary-like structure formation. We found that IL1β, IFNγ, and/or TNFα increased PGI2 secretion and PTGIR expression in pEETH cells. Iloprost (a PGI2 analogue) acting through PTGIR enhanced the transcript abundance of KDR, FGFR2, and ANGPT2 and increased proliferation of pEETH cells. This latter was mediated by PI3K and mTOR activation. In support, transfection of G1410 cells with siRNA targeting PGI2 synthase decreased pro-angiogenic gene expression and cell proliferation. Furthermore, iloprost accelerated the gap closure and promoted cell cycle progression. Intriguingly, the formation of capillary-like structures was inhibited but not completely blocked by iloprost. These findings point to a complex pleiotropic role of PGI2 in angiogenesis-related events in the porcine uterus.

Tumor-expressed B7-H3 promotes vasculogenic mimicry formation rather than angiogenesis in non-small cell lung cancer.

Vasculogenic mimicry (VM), an alternative microvascular circulation independent ofangiogenesis, is formed by aggressive cancer cells. Tumor-expressedB7-H3 has been reported to promote VM formation in hepatocellular carcinoma and modulate angiogenesis in breast cancer and colorectal cancer. However, its effects on VM generation and angiogenesis in non-small cell Lung cancer (NSCLC) remained to be elucidated. CRISPR/Cas9-mediated B7-H3 knockout (KO)was conducted in NSCLC A549 and H3255 cells. The expression of VM-related proteins, including vascular endothelial (VE)-cadherin and matrix metalloproteinase 14 (MMP14), and the secretion of vascular endothelial growth factor (VEGF) were measured by western blotting and chemiluminescence assay in both B7-H3 KO and mock-edited A549 and H3255 cells. To examine VM formation, a three-dimensional (3D)culture model was used for B7-H3 KO and mock A549 and H3255 cells. For in vivo analysis, xenograft mice models were established using B7-H3 KO and mock-edited A549 cells, and immunohistochemical (CD31) and histochemical (periodic acid-Schiff, PAS) double staining were performed to identify VM and endothelial vessels in tumor tissues. Finally, specific signaling inhibitors were used to analyze B7-H3-induced signaling pathway responsible for VE-cadherin and MMP14 expression and VM generation. Higher expression of B7-H3 was associated with a worse prognosis and moreadvanced T-category in NSCLC. CRISPR/Cas9-mediated B7-H3 KOin A549 and H3255 cells led to decreased expression of VE-cadherin and MMP14; however,the secretion of VEGFby the two cell lines remained unchanged. In the3D cell culture model, bothB7-H3 KO A549 and H3255cells showed a significant reduction in the formation of capillary-like tubular structures compared to mock-edited cells. In thein vivo xenograft model, mock-edited A549 cells formed excessive PAS+ CD31-VM channels, while B7-H3 KO restrained VM formation in the xenograft tumors. However, no significant differenceswere found in CD31+ endothelial vessels between xenografts formed by B7-H3 KO and mock-edited A549 cells. Finally, we analyzed the signaling pathway responsible for B7-H3-induced VM formation and found that selective inhibition of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) hyperactivation by LY294002 was associated with decreased expression of MMP14 and VE-cadherin, and in vitro VM formation by both A549 and H3255 cells. Tumor-expressed B7-H3 acts via PI3K/AKT signaling pathway to promote VM formation by NSCLC cells while bears no effects on angiogenesis in NSCLC.

Abstract 2654: Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein

Abstract Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2654.

Prokineticin 1 is a novel factor regulating porcine corpus luteum function

Prokineticin 1 (PROK1) is a pleiotropic factor secreted by endocrine glands; however, its role has not been studied in the corpus luteum (CL) during pregnancy in any species. The present study aimed to investigate the contribution of PROK1 in regulating processes related to porcine CL function and regression: steroidogenesis, luteal cell apoptosis and viability, and angiogenesis. The luteal expression of PROK1 was greater on Days 12 and 14 of pregnancy compared to Day 9. PROK1 protein expression during pregnancy increased gradually and peaked on Day 14, when it was also significantly higher than that on Day 14 of the estrous cycle. Prokineticin receptor 1 (PROKR1) mRNA abundance increased on Days 12 and 14 of pregnancy, whereas PROKR2 elevated on Day 14 of the estrous cycle. PROK1, acting via PROKR1, stimulated the expression of genes involved in progesterone synthesis, as well as progesterone secretion by luteal tissue. PROK1–PROKR1 signaling reduced apoptosis and increased the viability of luteal cells. PROK1 acting through PROKR1 stimulated angiogenesis by increasing capillary-like structure formation by luteal endothelial cells and elevating angiogenin gene expression and VEGFA secretion by luteal tissue. Our results indicate that PROK1 regulates processes vital for maintaining luteal function during early pregnancy and the mid-luteal phase.

Circular RNA hsa_circ_0000519 contributes to angiogenesis and tumor progression in hepatocellular carcinoma through the miR-1296/E2F7 axis.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Uncontrolled angiogenesis plays a critical role in hepatocellular tumor growth and metastasis. In this study, we aimed to investigate the effects of circular RNA hsa_circ_0000519 and the potential involvement of microRNA (miR)-1296 and E2F transcription factor 7 (E2F7) in HCC development. Hsa_circ_0000519 was highly expressed in HCC cells and hepatocellular tumor tissues, and correlated with poor prognosis of HCC patients. Knockdown of hsa_circ_0000519 significantly reduced HCC cell viability, suppressed cell proliferation, and induced cell cycle arrest in G0/G1. Downregulation of hsa_circ_0000519 also inhibited formation of capillary-like endothelial structures in vitro and impeded microvessel formation in mice bearing HCC tumors. The migration and invasive capacities of HCC cells were markedly reduced by hsa_circ_0000519 knockdown. Hsa_circ_0000519 possessed a binding site for microRNA (miR)-1296. Upregulation of hsa_circ_0000519 significantly decreased the miR-1296 expression in both HCC cells and mouse xenografts. Furthermore, E2F7 was a target of miR-1296. Hsa_circ_0000519 positively regulated E2F7 via acting as a miR-1296 sponge. Upregulation of E2F7 abolished the inhibitory effects of hsa_circ_0000519 knockdown on HCC cell proliferation and angiogenesis. In conclusion, hsa_circ_0000519 promoted tumor progression and angiogenesis in HCC through the miR-1296/E2F7 axis. These data suggest the potential clinical application of hsa_circ_0000519 in HCC treatment.

Application of thermosensitive-hydrogel combined with dental pulp stem cells on the injured fallopian tube mucosa in an animal model.

Objectives: Fallopian tube (FT) injury is an important factor that can lead to tubal infertility. Stem-cell-based therapy shows great potential for the treatment of injured fallopian tube. However, little research has shown that mesenchymal stem cells (MSCs) can be used to treat fallopian tube damage by in situ injection. In this study, we in situ transplanted PF127 hydrogel encapsulating dental pulp stem cells (DPSCs) into the injured sites to promote the repair and regeneration of fallopian tube injury. Materials and methods: The properties of dental pulp stem cells were evaluated by flow cytometry, immunofluorescence analysis, and multi-differentiation detection. The immunomodulatory and angiogenic characteristics of dental pulp stem cells were analyzed on the basis of the detection of inflammatory factor expression and the formation of capillary-like structures, respectively. The biocompatibility of PF127 hydrogel was evaluated by using Live/Dead and CCK-8 assays. The effects of PF127 hydrogel containing dental pulp stem cells on the repair and regeneration of fallopian tube injury were evaluated by histological analysis [e.g., hematoxylin and eosin (H&E) and Masson's trichrome staining, TUNEL staining, immunofluorescence staining, and immunohistochemistry], Enzyme-linked immunosorbent assay (ELISA), and RT-PCR detections. Results: Dental pulp stem cells had MSC-like characteristics and great immunomodulatory and angiogenic properties. PF127 hydrogel had a thermosensitive feature and great cytocompatibility with dental pulp stem cells. In addition, our results indicated that PF127 hydrogel containing dental pulp stem cells could promote the repair and regeneration of fallopian tube damage by inhibiting cell apoptosis, stimulating the secretion of angiogenic factors, promoting cell proliferation, modulating the secretion of inflammatory factors, and restoring the secretion of epithelial cells. Conclusion: In this study, our results reported that in situ injection of PF127 hydrogel encapsulating dental pulp stem cells into the injured sites could provide an attractive strategy for the future treatment of fallopian tube injury in clinical settings.

RNF213 Loss-of-Function Promotes Angiogenesis of Cerebral Microvascular Endothelial Cells in a Cellular State Dependent Manner.

Enhanced and aberrant angiogenesis is one of the main features of Moyamoya disease (MMD) pathogenesis. The ring finger protein 213 (RNF213) and the variant p.R4810K have been linked with higher risks of MMD and intracranial arterial occlusion development in east Asian populations. The role of RNF213 in diverse aspects of the angiogenic process, such as proliferation, migration and capillary-like formation, is well-known but has been difficult to model in vitro. To evaluate the effect of the RNF213 MMD-associated gene on the angiogenic activity, we have generated RNF213 knockout in human cerebral microvascular endothelial cells (hCMEC/D3-RNF213-/-) using the CRISPR-Cas9 system. Matrigel-based assay and a tri-dimensional (3D) vascularized model using the self-assembly approach of tissue engineering were used to assess the formation of capillary-like structures. Quite interestingly, this innovative in vitro model of MMD recapitulated, for the first time, disease-associated pathophysiological features such as significant increase in angiogenesis in confluent endothelial cells devoid of RNF213 expression. These cells, grown to confluence, also showed a pro-angiogenic signature, i.e., increased secretion of soluble pro-angiogenic factors, that could be eventually used as biomarkers. Interestingly, we demonstrated that that these MMD-associated phenotypes are dependent of the cellular state, as only noted in confluent cells and not in proliferative RNF213-deficient cells.

CircANKRD12 Is Induced in Endothelial Cell Response to Oxidative Stress.

Redox imbalance of the endothelial cells (ECs) plays a causative role in a variety of cardiovascular diseases. In order to better understand the molecular mechanisms of the endothelial response to oxidative stress, the involvement of circular RNAs (circRNAs) was investigated. CircRNAs are RNA species generated by a "back-splicing" event, which is the covalent linking of the 3'- and 5'-ends of exons. Bioinformatics analysis of the transcriptomic landscape of human ECs exposed to H2O2 allowed us to identify a subset of highly expressed circRNAs compared to their linear RNA counterparts, suggesting a potential biological relevance. Specifically, circular Ankyrin Repeat Domain 12 (circANKRD12), derived from the junction of exon 2 and exon 8 of the ANKRD12 gene (hsa_circ_0000826), was significantly induced in H2O2-treated ECs. Conversely, the linear RNA isoform of ANKRD12 was not modulated. An increased circular-to-linear ratio of ANKRD12 was also observed in cultured ECs exposed to hypoxia and in skeletal muscle biopsies of patients affected by critical limb ischemia (CLI), two conditions associated with redox imbalance and oxidative stress. The functional relevance of circANKRD12 was shown by the inhibition of EC formation of capillary-like structures upon silencing of the circular but not of the linear isoform of ANKRD12. Bioinformatics analysis of the circANKRD12-miRNA-mRNA regulatory network in H2O2-treated ECs identified the enrichment of the p53 and Foxo signaling pathways, both crucial in the cellular response to redox imbalance. In keeping with the antiproliferative action of the p53 pathway, circANKRD12 silencing inhibited EC proliferation. In conclusion, this study indicates circANKRD12 as an important player in ECs exposed to oxidative stress.

Possible involvement of pro-angiogenic activities in the traditional use of the stembark of Oenocarpus bacaba Mart. (Arecaceae) for wound healing in the Republic of Suriname (South America)

Angiogenesis is an important part of the wound healing process. In this study, the aqueous extracts from seven plant species that are used for wound care in the Republic of Suriname (South America), were evaluated at sub-toxic concentrations for their stimulatory effects on the closure of scratch-wounds in cultured human umbilical vein endothelial cells (HUVECs), the formation of capillary-like structures by these cells, and the growth of sub-intestinal blood vessels in developing Tg (fli1a: EGFP) y1/+ zebrafish embryos. Sub-toxic extract concentrations were about one-third of IC50 values in HUVECs which were established using a sulforhodamine B assay after a 3-day exposure period. Data were expressed relatively to those found with untreated controls and considered statistically significantly different from each other when p values < 0.05 (ANOVA). When compared to untreated controls, the Oenocarpus bacaba stembark extract decreased HUVEC scratch-wound areas by about 30%; increased tube length, number of branching points, and number of loops formed by HUVECs by 50-70%, and increased total sub-intestinal blood vessel length in the zebrafish embryos by about 30%. The extracts from Morinda citrifolia (leaf), Luffa acutangula (fruit juice), Momordica charantia (leaf), Psidium guajava (leaf), Cecropia peltata (branch tops), and Spondias mombin (leaf) had no statistically significant effect on any of these variables. These observations suggest that the O. bacaba sample, unlike the other samples, possessed pro-angiogenic properties which may be involved in its beneficial effects in wound healing. Future studies should more elaborately evaluate these plants in order to definitely establish their therapeutic value in wound healing.

Integrin-specific hydrogels for growth factor-free vasculogenesis

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvβ3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvβ3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvβ3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.

Angiogenic properties of glial progenitor cells derived from human induced pluripotent stem cells

Diseases associated with impaired blood supply to the brain ranks second term of mortality in the world, losing the place only to coronary heart disease. The incidence of this disease in the world remains high and increasing significantly with the age. The recent years special attention has been paid to the search for new methods of therapy for ischemic diseases, such as study of angiogenic properties of stem cells and their conditioned medium. The aim of this work is studying the angiogenic properties of glial progenitor cells derived from human induced pluripotent stem cells. The study was carried out by testing the proliferative activity, mobility, migration of endothelial cells line EA.hy926 under the influence of glial progenitor cells and their conditioned medium. Also the research was conducted by ability to formation of the tubular and capillary-like structure by cells line EA.hy926 by modeling angiogenesis in the basement membrane matrix in vitro. The conditioned medium obtained by glial progenitor cells at concentrations of total protein 1 and 5 g/ml has a positive influence on the proliferative activity and mobility of the endothelial cells line EA.hy926. At the same time it does not accelerate the formation of the primary tubular and capillary-like structure by the modeling angiogenesis in the basement membrane matrix in vitro. But glial progenitor cells contribute to the formation of tubular and capillary-like structure due to contact-dependent signaling between the two cell types. The primary formed tubular structure had a long processes and large branch points under co cultivation with glial progenitor cells. Sprouting centers also had long and more convoluted processes and large cell clusters during the formation of a capillary-like structure. The glial progenitor cells and their conditioned medium had a positive effect on endothelial cell migration. This effect probably indicated by the production of substances by glial progenitor cells which was chemoattractants for endothelial cells line EA.hy926.