Endothelial Cell Morphogenesis Research Articles

THE SUCCESS OF ND-YAG LASER IN THE TREATMENT OF VENOUS MALFORMATIONS OF THE ORAL MUCOSA ABOUT TWO CASES

Venous malformations are the result of errors in endothelial cell morphogenesis, causing disorganized angiogenesis and intimal smooth muscle proliferation. (1) This results in the formation of dilated networks of venous lakes that are hemodynamically non-functional. (1,2) Depending on location, they may go unnoticed for years prior to presentation, and range from asymptomatic superficial varicosities to large deforming craniofacial lesions. The management of venous malformation will depend on the localization and expansion of the malformation, and there are multiple therapeutic approaches: sclerotherapy, surgery, and medical management. Lasers, another modality for treating VMs. we report two casesof success with the Nd Yag laser in the treatment ofsuperficialvenous malformations the oral mucosa.

Abstract 7298: A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels

Abstract Introduction: Extra virgin olive oil is a basic component of the Mediterranean diet; it contains several molecules with antioxidant, anti-inflammatory, anti-angiogenic and pro-apoptotic properties against a variety of cancers and influencing cellular metabolism too. Olive mill waste water (OMWW), a liquid residue produced in vast amounts during olive oil extraction, represents an environmental issue but on the other side it is a rich source of phytochemicals with potential health benefits. Particularly, OMWWs are rich in different phenolic compounds, that show promise in preventing the development of several disease disorders, including cancer. We have assessed the anti-angiogenic and anti-tumor properties exerted by a nutraceutical from purified OMWW extract, in in vitro and in vivo models, and evaluated its effect in a human pilot study upon informed consent. Experimental procedures: OMWWs ability to affect cell proliferation and survival on different cancer cell lines was assessed by MTT. OMWWs interference with human umbilical endothelial vein cells (HUVECs) tube formation, migration and invasive capacities was studied by endothelial cell morphogenesis and migration assays. The inhibition of angiogenesis and tumor cell growth was investigated by the matrigel assay and tumour xenograft. To evaluate contribution of potential metabolomic biomarkers, SANIST (surface-activated chemical ionization-electrospray-NIST technology) mass spectrometric data-based platform has been employed on sera from a pilot study (Aiello et al, in preparation). Results: OMWW extract shown a potent inhibitory effect inhibiting cells proliferation of both cancer cell lines and HUVECs.OMWW inhibited several molecular targets expression, including IL8, Angiogenin, mTOR, VEGF, CXCR4, CXCL12 and CXCL8, in tumor cells and in TNF-α-stimulated HUVEC. We nalyzed sera from volunteers, after nutraceutical intervention with Oliphenoliaand we assessed several biological parameters. Endogenous vitamin D was significantly increased, as well as albumin, direct bilirubin, and calcium. We also observed glycemia and potassium decrease after Oliphenolia assumption. A previously unknow form of Vitamin D was also detected by mass spectrometry. Conclusions: OMWW extract has shown a stronger anti-angiogenic and anti-tumor properties, with potential chemo-preventive potentials. Results of human pilot study shows Oliphenolia as a promising and interesting natural dietary supplement, rich in polyphenol compounds, that can exert anti-inflammatory and antioxidant activities. Acknowledgements: This study was supported by a donation from the Fattoria La Vialla di Gianni, Antonio e Bandino Lo Franco - SAS (Arezzo, Italy). We are grateful to Professor Anna Aiello, and Professor Calogero Caruso, Palermo University for the pilot study. Citation Format: Adriana Albini, Luana Calabrone, Francesca Albini, Paola Corradino, Simone Cristoni, Douglas M. Noonan. A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7298.

Abstract C116: Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C116.

Elucidating the Morphogenic and Signaling Roles of Defined Growth Factors Controlling Human Endothelial Cell Lumen Formation Versus Sprouting Behavior

Five growth factors [ie, insulin, fibroblast growth factor-2 (FGF-2), stem cell factor, IL-3, and stromal-derived factor 1α] in combination are necessary for human endothelial cells (ECs) to undergo tube morphogenesis, a process requiring both lumen formation and sprouting behavior. This study investigated why these factors are required by subdividing the factors into 4 separate groups: insulin-only, insulinand FGF-2, no FGF-2 (all factors but without FGF-2), and all factors. The study found that the insulin-only condition failed to support EC morphogenesis or survival, the insulinand FGF-2 condition supported primarily EC lumen formation, and the no FGF-2 condition supported EC sprouting behavior. By comparison, the all-factors condition more strongly stimulated both EC lumen formation and sprouting behavior, and signaling analysis revealed prolonged stimulation of multiple promorphogenic signals coupled with inhibition of proregressive signals. Pharmacologic inhibition of Jak kinases more selectively blocked EC sprouting behavior, whereas inhibition of Raf, phosphatidylinositol 3-kinase, and Akt kinases showed selective blockade of lumen formation. Inhibition of Src family kinases and Notch led to increased sprouting coupled to decreased lumen formation, whereas inhibition of Pak, Mek, and mammalian target of rapamycin kinases blocked both sprouting and lumen formation. These findings reveal novel downstream biological and signaling activities of defined factors that are required for the assembly of human EC-lined capillary tube networks.

Abstract 124: Renin Cell Identity And Blood Pressure Homeostasis: Exploring The Role Of Endothelial Cell Cross-talk

Our previous studies using an Aortic Coarctation (AoCo) mouse model identified that renin-expressing juxta-glomerular (JG) cells respond to changes in perfusion pressure and Lamin A/C as a component of the renin cell baroreceptor. However, as the mechanisms for transmitting the extracellular pressure to the JG cells are unclear, we hypothesized that the endothelial cells (ECs) act as transducers of signals to the JG cells. Therefore in the current study, we aim 1) to identify the concurrent transcriptomic and epigenomic changes in JG cells and ECs in response to the changes in the perfusion pressure by single-nuclei multiomics approach 2) to define the contribution of EC-specific Lamin A/C (Lmna) in the EC-JG-cells cross-talk. We performed the AoCo surgeries on 3 months (M) old VE-Cad-Cre;Foxd1-Cre;mTmG mice where GFP is expressed simultaneously in Foxd1 and EC-lineage cells. We compared the single-cell gene expression and chromatin accessibility between the left (LK) and the right kidneys (RK) at 72h post-surgeries. Our study revealed: 1) JG cells increased significantly in the LK along with renin gene expression 2) ECs significantly increased by 10.73% in LK with a higher expression for genes promoting EC proliferation, morphogenesis, and immune response 3) JGs of the LK showed enrichment for the motifs Mbd1, Smad1, Smad5 indicating epigenetic changes promoting transcriptional regulation and TgfB1 signaling. Enrichment of Mybl1, Myb, and E2f2 motifs in the ECs of the LK suggests positive regulation of EC proliferation in response to low blood pressure (BP). Furthermore, deletion of the Lmna gene in the EC-lineage affected the BP responses in 4M old Lmna fl/fl ;VE-Cad.Cre mice. The difference between the carotid and femoral mean BP (MBP) measurements was more prominent in the wild type (WT) compared to the mutants at 72h post-AoCo [WT (n=7), Carotid MBP: 69.16±2.24 Femoral MBP: 63.28±2.33, P<0.00001 Mutants (n=4); Carotid MBP: 75.98±3.06 Femoral MBP: 69.75±3.10, P<0.05)]. Our current study shows that ECs undergo transcriptional and epigenetic alterations in response to changes in perfusion pressure. Deletion of Lmna in ECs reduces the differences in the BP between LK and RK, suggesting that it is important for sensing the changes in extracellular pressure.

Surface-functionalized Zinc MOFs delivering zinc ion and hydrogen sulfide as tailored anti-hindlimb ischemic nanomedicine

Therapeutic angiogenesis is one of the most promising solutions to the clinical dilemma of critical limb ischemia (CLI). Promoting the morphogenesis of endothelial cells (ECs) and normalizing the microenvironment are the right pathways for angiogenesis, but existing strategies are limited by inefficiency, complexity and high cost. Oriented by clinical needs and engineering applications, an innovative nano-system was developed from bridging facile/cost-effective metal-organic frameworks (MOFs) for delivering bioactive zinc ion and versatile hydrogen sulfide (H2S). These MOFs were surface-modified with a multi-functional layer and evaluated as a potential tailored anti-hindlimb ischemic nanomedicine. As a holistic strategy, the combination of zinc ion and H2S generated from zinc MOFs (Zn-MOFs) and diallyl trisulfide (DATS) can integrally modulate EC behavior and microenvironment for more effective functional rehabilitation of ischemic limb. Specifically, zinc ion and H2S together amplified the pro-angiogenic effect on ECs, while H2S also alleviated post-ischemic oxidative stress and inflammation by inducing pro-healing and anti-inflammatory M2-type macrophages. Furthermore, surface-engineering of MOFs reduced the toxicity of nanomedicine and improved its utilization in intramuscular injection. Overall, the nano-system not only enables safe and effective H2S administration, but also presents a new potential option for CLI clinical therapy as an efficacious/facile/cost-effective nanomedicine.

Mechanomorphological Guidance of Colloidal Gel Regulates Cell Morphogenesis.

Microstructural morphology of the extracellular matrix guides the organization of cells in 3D. However, current biomaterials-based matrices cannot provide distinct spatial cues through their microstructural morphology due to design constraints. To address this, colloidal gels are developed as 3D matrices with distinct microstructure by aggregating ionic polyurethane colloids via electrostatic screening. Due to the defined orientation of interconnected particles, positively charged colloids form extended strands resulting in a dense microstructure whereas negatively charged colloids form compact aggregates with localized large voids. Chondrogenesis of human mesenchymal stem cells (MSCs) and endothelial morphogenesis of human endothelial cells (ECs) are examined in these colloidal gels. MSCs show enhanced chondrogenic response in dense colloidal gel due to their spatial organization achieved by balancing the cell-cell and cell-matrix interactions compared to porous gels where cells are mainly clustered. ECs tend to form relatively elongated cellular networks in dense colloidal gel compared to porous gels. Additionally, the role of matrix stiffness and viscoelasticity in the morphogenesis of MSCs and ECs are analyzed with respect to microstructural morphology. Overall, these results demonstrate that colloidal gels can provide spatial cues through their microstructural morphology and in correlation with matrix mechanics for cell morphogenesis.

"Intercellular Mass Transport" Mimic Enables ASO Entry Completely into the Cell Nucleus for Enhanced Ischemia Therapy.

Currently, the development of a new therapeutic technology is focused on antisense oligonucleotides (ASOs), where ASOs are used to complementarily pair with DNA, messenger RNA, or long noncoding RNA (lncRNA) to regulate the cell behavior by inhibiting the target gene expression. However, the targeted regulation toward nuclear genes still faces great challenges in ASO delivery for clinical applications, i.e., two essential criteria (high nuclear entry and delivery vehicle safety/simplification) generally compromise each other and are not simultaneously satisfactory. Herein, for the first time, inspired by "intercellular-mass-transport", we report an important discovery that the cell membrane of endothelial cells (ECs) serving as the biointerface enables ASOs to rapidly and completely enter the EC nucleus. Thereby, we innovatively fabricate a nanosystem only by sequential self-assembly of natural/off-the-shelf biomaterials to well overcome the above-mentioned contradiction. The efficacy is strikingly superior to that of the previous delivery vehicles. Furthermore, our technology is applied to successfully silence lncRNA MEG3 in the EC nucleus, significantly augmenting EC morphogenesis. More importantly, this nanosystem is applicable for in vivo intramuscular injection to enhance the therapeutic outcome in a critical limb ischemia mouse model. This work brings a new hope for the technological innovation of ASO nuclear delivery and opens a new avenue to explore natural/off-the-shelf materials for cargo delivery into subcellular compartments.

Dynamically regulated focal adhesions coordinate endothelial cell remodelling in developing vasculature.

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.

Delivery of VEGF and delta-like 4 to synergistically regenerate capillaries and arterioles in ischemic limbs

Vascularization of the poorly vascularized limbs affected by critical limb ischemia (CLI) is necessary to salvage the limbs and avoid amputation. Effective vascularization requires forming not only capillaries, but also arterioles and vessel branching. These processes rely on the survival, migration and morphogenesis of endothelial cells in the ischemic limbs. Yet endothelial cell functions are impaired by the upregulated TGFβ. Herein, we developed an injectable hydrogel-based drug release system capable of delivering both VEGF and Dll4 to synergistically restore endothelial cellular functions, leading to accelerated formation of capillaries, arterioles and vessel branching. In vitro, the Dll4 and VEGF synergistically promoted the human arterial endothelial cell (HAEC) survival, migration, and formation of filopodial structure, lumens, and branches under the elevated TGFβ1 condition mimicking that of the ischemic limbs. The synergistic effect was resulted from activating VEGFR2, Notch-1 and Erk1/2 signaling pathways. After delivering the Dll4 and VEGF via an injectable and thermosensitive hydrogel to the ischemic mouse hindlimbs, 95% of blood perfusion was restored at day 14, significantly higher than delivery of Dll4 or VEGF only. The released Dll4 and VEGF significantly increased density of capillaries and arterioles, vessel branching point density, and proliferating cell density. Besides, the delivery of Dll4 and VEGF stimulated skeletal muscle regeneration and improved muscle function. Overall, the developed hydrogel-based Dll4 and VEGF delivery system promoted ischemic limb vascularization and muscle regeneration. Statement of significanceEffective vascularization of the poorly vascularized limbs affected by critical limb ischemia (CLI) requires forming not only capillaries, but also arterioles and vessel branching. These processes rely on the survival, migration and morphogenesis of endothelial cells. Yet endothelial cell functions are impaired by the upregulated TGFβ in the ischemic limbs. Herein, we developed an injectable hydrogel-based drug release system capable of delivering both VEGF and Dll4 to synergistically restore endothelial cell functions, leading to accelerated formation of capillaries, arterioles and vessel branching.

Superlow Dosage of Intrinsically Bioactive Zinc Metal-Organic Frameworks to Modulate Endothelial Cell Morphogenesis and Significantly Rescue Ischemic Disease.

Despite long-term efforts for ischemia therapy, proangiogenic drugs hardly satisfy therapy/safety/cost/mass production multiple evaluations and meanwhile with a desire to minimize dosages, thereby clinical applications have been severely hampered. Recently, metal ion-based therapy has emerged as an effective strategy. Herein, intrinsically bioactive Zn metal-organic frameworks (MOFs) were explored by bridging the dual superiorities of proangiogenic Zn2+ and facile/cost-effective/scalable MOFs. Zn-MOFs could enhance the morphogenesis of vascular endothelial cells (ECs) via the PI3K/Akt/eNOS pathway. However, high dosage is inevitable and Zn-MOFs suffer from insolubility and low stability, which lead to the bioaccumulation of Zn-MOFs and seriously potential toxicity risks. To alleviate this, it is required to decrease the dosage, but this can be entrapped into the dosage/therapy/safety contradiction and disappointing therapy effect. To address these challenges, the bioavailability of Zn-MOFs is urgent to improve for the minimization of dosage and significant therapy/safety. The mitochondrial respiratory chain is Zn2+ active, which inspired us to codecorate EC-targeted and mitochondria-localizing-sequence peptides onto Zn-MOF surfaces. Interestingly, after codecoration, a 100-fold reduced dosage acquired equally powerful vascularization, and the superlow dosage significantly rescued ischemia (4.4 μg kg-1, about one order of magnitude lower than the published minimal value). Additionally, no obvious muscle injury was found after treatment. Potential toxicity risks were alleviated, benefiting from the superlow dosage. This advanced drug simultaneously satisfied comprehensive evaluations and dosage minimization. This work utilizes engineering thought to rationally design "all-around" bioactive MOFs and is expected to be applied for ischemia treatment.

Human iPSC-Derived Vascular Smooth Muscle Cells in a Fibronectin Functionalized Collagen Hydrogel Augment Endothelial Cell Morphogenesis.

Tissue-engineered constructs have immense potential as autologous grafts for wound healing. Despite the rapid advancement in fabrication technology, the major limitation is controlling angiogenesis within these constructs to form a vascular network. Here, we aimed to develop a 3D hydrogel that can regulate angiogenesis. We tested the effect of fibronectin and vascular smooth muscle cells derived from human induced pluripotent stem cells (hiPSC-VSMC) on the morphogenesis of endothelial cells. The results demonstrate that fibronectin increases the number of EC networks. However, hiPSC-VSMC in the hydrogel further substantiated the number and size of EC networks by vascular endothelial growth factor and basic fibroblast growth factor secretion. A mechanistic study shows that blocking αvβ3 integrin signaling between hiPSC-VSMC and fibronectin impacts the EC network formation via reduced cell viability and proangiogenic growth factor secretion. Collectively, this study set forth initial design criteria in developing an improved pre-vascularized construct.

Ubiquitin-conjugating enzyme V variant 1 enables cellular responses toward fibroblast growth factor signaling in endothelium.

Ubiquitination has been shown to provide an essential regulatory role in modulating the duration and amplitude of the signaling activity in angiogenesis. While successive enzymatic reactions mediated by three distinct types of enzymes commonly known as E1, E2, and E3 are required for ubiquitination, the role of E3s which govern the final step of ubiquitination has been extensively analyzed in angiogenesis. In contrast, the role of E2s, which determine the context and functional consequences of ubiquitination, remains largely unknown with respect to angiogenesis. To better elucidate the role of E2s in modulating endothelial behaviors during angiogenesis, we first systematically analyze the expression pattern of E2s in endothelial cells (ECs) using previously published scRNA-seq data and identify ubiquitin-conjugating enzyme variant 1 (UBE2V1), an unconventional E2 without innate catalytic activity, as one of the most abundantly expressed E2s in ECs. While ubiquitously expressed in diverse cell types, abrogation of UBE2V1significantly impairs proliferation and viability of human umbilical vein endothelial cells (HUVECs) without affecting other cell types, suggesting that UBE2V1 is likely to possess nonredundant functions in ECs. Consistent with this idea, UBE2V1 appears to be critical for morphogenesis and migration of ECs during angiogenesis. Interestingly, we find that UBE2V1 is essential for fibroblast growth factor 2 (FGF2)-induced angiogenesis, but appears to have minor effects on vascular endothelial growth factor-A-induced angiogenesis in vitro as well as in vivo. Therefore, it seems that UBE2V1 could enable ECs to distinguish two related yet distinct angiogenic cues. Mechanistically, we show that UBE2V1 promotes ubiquitination of MEK kinase 1, a key mediator of FGF2signaling, to enhance phosphorylation of extracellular signal-regulated kinase 1/2 in HUVECs. Taken together, our results illustrate the unique role of UBE2V1 as a key modulator for angiogenic behaviors in ECs.

LncRNA-mRNA Co-expression Profiles Relative to Vascular Remodeling in Moyamoya Patients Without RNF213 Mutation

Moyamoya disease (MMD) is an idiopathic cerebrovascular disease with unknown etiology. Long noncoding RNA (lncRNA) and messenger RNA (mRNA) profiles in MMD remain unknown. In this current study, we aim to investigate lncRNA-mRNA co-expression pattern and their biological functions in superficial temporal artery (STA) of MMD. STA of 3 MMD patients without RNF213 mutation and 3 age-matched controls were obtained for transcriptomic RNA sequencing. Bioinformatics analysis was performed to investigate their molecular functions and interactions. Then, differentially expressed genes relative to vascular remodeling were further validated by quantitative real-time polymerase chain reaction and immunofluorescence. WNT5A functions were tested by tube formation assay and wound scratching assay in human microvascular endothelial cells (HMECs). We detected 6235 different lncRNAs and 2065 different mRNAs from the RNA-sequencing between MMD patients and controls (P < 0.05; fold change >2.0). Gene ontology showed that altered mRNAs were enriched for endothelial cell morphogenesis and positive regulation of angiogenesis, which were closely related with vascular remodeling. We then searched 76 altered genes related with vascular remodeling and applied Kyoto Encyclopedia of Genes and Genomes analysis. Integrated analysis of lncRNA-TF-mRNA co-expression networks and gene verifications indicated that molecular including WNT5A, TEK, and GATA2 may contribute to the vascular malformation of MMD. Overexpression of WNT5A in HMECs promoted tube formation and cell migration. In MMD patients, genes related to vascular remodeling including WNT5A and their regulators were aberrantly disrupted. These results will help elucidate the complicated pathogenic mechanism of MMD and develop potential therapeutic targets facilitating MMD angiogenesis in the future.

Gene expression profiles of two testicular somatic cell lines respond differently to 4-nitrophenol mediating vary reproductive toxicity

4-Nitrophenol (PNP) has been extensively used in manufacturing for several decades. Its toxic effects on the male reproductive system have been reported, but the underlying mechanisms remain unclear. In this study, we utilized two testicular somatic cell lines (TM3 and TM4 cells) to explore the possible toxic effects of PNP on the male reproductive system. The activity of the cells after exposure to different doses of PNP (0.01, 0.1, 1, 10 and 100 μM) was evaluated. PNP treatment at 10 μM significantly inhibited cell viability, and 10 μM PNP was thus selected for subsequent experiments. Although PNP (10 μM) inhibited cell proliferation, promoted cell apoptosis, and changed the cell cycle distribution and ultrastructure in both types of cells, these effects were more significant in the TM4 cells. In addition, an Agilent mouse mRNA array was used to identify the gene expression differences between the control and PNP (10 μM) exposed TM3 and TM4 cells. The microarray analysis identified 67 and 1372 differentially expressed genes mainly concentrated in endothelial cell morphogenesis and anatomical structure development in TM3 cells and associated with cardiovascular system development and circulatory system development in TM4 cells. Moreover, a pathway analysis revealed that PNP not only predominately affected meiotic recombination and meiosis in TM3 cells, but also influenced axon guidance and developmental biology in TM4 cells. These results suggest that TM3 and TM4 cells exhibit different responses to PNP, which might mediate different toxic mechanisms.

Endothelial Cell Morphogenesis and Capillary-like Network Induced by Soluble and Bound VEGF in a Definite Biogel Composed of Collagen and Fibronectin

In vitro culture of endothelial cells to form capillary-like networks is essential in tissue engineering. Vascular endothelial growth factor (VEGF) is one of the primary signal proteins stimulating blood vessel formation. This growth factor can be soluble in the medium or protein-bound to the substrate. However, less attention has been paid to distinguishing the specific stimulations by soluble and bound VEGF. We conducted a series of experiments to explore the respective effects of these two VEGF forms. An in-house synthesized biogel comprising a definite concentration of collagen and fibronectin was designed to cultivate human umbilical vein endothelial cells to form the capillary-like network. Collagen served as the primary substrate for cell attachment. Fibronectin provided the surface to bind soluble VEGF in the culture medium to create the bound VEGF. The experiment of adding VEGF-blocking-peptide was conducted to prevent the formation of VEGF bound to the fibronectin domains, to distinguish the respective effects of the soluble and bound VEGF. With the in-house biogel of definite components, we were able to clarify the different roles of soluble and bound VEGF. The results indicated that the soluble VEGF promptly induced the cells to change from round to elongated shape, which contributed to forming network cords. Simultaneously, the bound VEGF provided long-term stimulation, causing the cells to migrate and differentiate into the final capillary-like network.

Three-dimensional microengineered vascularised endometrium-on-a-chip.

STUDY QUESTIONCan we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model?SUMMARY ANSWEROur representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner.WHAT IS KNOWN ALREADYOrgan-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems.STUDY DESIGN, SIZE, DURATIONThe device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5–6 days (total ∼ 14 days) for the purpose of each experiment.PARTICIPANTS/MATERIALS, SETTING, METHODSMicrofluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients.MAIN RESULTS AND THE ROLE OF CHANCEWe established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells.WIDER IMPLICATIONS OF THE FINDINGSOur 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner.STUDY FUNDING/COMPETING INTEREST(S)This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.

Pkd1 and Wnt5a genetically interact to control lymphatic vascular morphogenesis in mice.

Lymphatic vascular development is regulated by well-characterized signaling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity, and morphogenesis. Canonical and non-canonical WNT signaling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signaling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1;Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signaling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signaling during the formation of lymphatic vascular networks.

Assessing the Angiogenic Efficacy of Pleiotrophin Released from Injectable Heparin-Alginate Gels.

With this work, we design alginate-based hydrogels for therapeutically directing revascularization and repair processes in vivo. We immobilize pleiotrophin (PTN) in injectable hydrogel formulations as the target factor to stimulate proangiogenic responses in endothelial cells. The optimized heparin-alginate/chitosan hydrogels, produced by internal crosslinking with calcium carbonate, show good biocompatibility and injectability and allow controlling the release of immobilized proteins in the subcutaneous tissue over a period of 7 days. In vitro assays, performed with translational human induced pluripotent stem cell-derived endothelial cells, and the in vivo Matrigel plug assay are conducted to demonstrate the angiogenic effects of PTN on endothelial cells. Our results indicate that PTN stimulates endothelial cell morphogenesis in vitro and the migration of endothelial cells and macrophages as soon as 4 days after injections of the developed hydrogels, promoting the formation of structures similar to the healthy granulation tissue, which is an indicator of healing in ischemic wounds. These studies provide the rationale for further investigating this novel therapeutic for pursuing increased vascular density for efficient regeneration of ischemic tissues, by leveraging the host endothelial cell population to initiate angiogenic and reparative processes in vivo. Impact statement Localized, sustained, and controlled delivery of angiogenic factors is crucial for enabling the formation of novel vascular networks in ischemic tissues. This study describes the development of an injectable heparin-alginate/collagen hydrogel for controlling the in vivo release and bioactivity of pleiotrophin (PTN), a heparin-binding factor with significant angiogenic activity. We demonstrate that PTN promotes angiogenesis in an in vitro model of hypoxia and in preclinical subcutaneous models. These results advance our understanding of PTN function in guiding therapeutic angiogenesis and are critical to inform the development of novel translational strategies for ischemic tissue repair and regeneration.

Prevascularized hydrogels with mature vascular networks promote the regeneration of critical-size calvarial bone defects in vivo.

Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors.