Vascular Network Formation Research Articles

Strontium and simvastatin dual loaded hydroxyapatite microsphere reinforced poly(ε-caprolactone) scaffolds promote vascularized bone regeneration.

The promotion of vascular network formation in the early stages of implantation is considered a prerequisite for successful functional bone regeneration. In this study, we successfully constructed 3D printed scaffolds with strong mechanical strength and a controllable pore structure that can sustainably release strontium (Sr) ions and simvastatin (SIM) for up to 28 days by incorporation of Sr2+ and SIM-loaded hydroxyapatite microspheres (MHA) into a poly(ε-caprolactone) (PCL) matrix. In vitro cell experiments showed that Sr-doped scaffolds were beneficial to the proliferation and osteogenic differentiation of bone mesenchymal stem cells (BMSCs), an appropriate dose of SIM was beneficial to cell proliferation and angiogenesis, and a high dose of SIM was cytotoxic. The Sr- and SIM-dual-loaded scaffolds with an appropriate dose significantly induced osteogenic differentiation of BMSCs and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro and promoted vascular network and functional bone formation in vivo. Ribose nucleic acid (RNA) sequencing analysis suggested that the mechanism of promotion of vascularized bone regeneration by fabricated scaffolds is that dual-loaded Sr2+ and SIM can upregulate osteogenic and vasculogenic-related genes and downregulate osteoclast-related genes, which is beneficial for vascular and new bone regeneration. The 3D printed composite scaffolds loaded with high-stability and low-cost inorganic Sr2+ ions and SIM small-molecule drugs hold great promise in the field of promoting vascularized bone regeneration.

Patterns of development and formation of the fetal central nervous system integrative function in the antenatal period

The development of the fetal central nervous system and the formation of its integrative functions have been studied for a long time. In the middle of the 20th century, researchers paid attention to structural changes and in the 1980s to the sequence of formation of functional relationships in the fetal body and the possibilities of their assessment. Further development of technology (accumulation of knowledge in the field of embryology, better resolution of ultrasound diagnostic devices, introduction and improvement of magnetic resonance imaging methods) allowed for not only receiving more detailed data on structural patterns in the fetal brain during pregnancy, but also presenting new opportunities for expanding knowledge about its functional condition. The review is devoted to the generalization of knowledge about the development of the fetal central nervous system, the brain vascular network formation and the brain circulation, as well as possibilities of assessing the formation of the fetal central nervous system integrative function during the entire period of pregnancy.

Bioengineering for vascularization: Trends and directions of photocrosslinkable gelatin methacrylate hydrogels.

Gelatin methacrylate (GelMA) hydrogels have been widely used in various biomedical applications, especially in tissue engineering and regenerative medicine, for their excellent biocompatibility and biodegradability. GelMA crosslinks to form a hydrogel when exposed to light irradiation in the presence of photoinitiators. The mechanical characteristics of GelMA hydrogels are highly tunable by changing the crosslinking conditions, including the GelMA polymer concentration, degree of methacrylation, light wavelength and intensity, and light exposure time et al. In this regard, GelMA hydrogels can be adjusted to closely resemble the native extracellular matrix (ECM) properties for the specific functions of target tissues. Therefore, this review focuses on the applications of GelMA hydrogels for bioengineering human vascular networks in vitro and in vivo. Since most tissues require vasculature to provide nutrients and oxygen to individual cells, timely vascularization is critical to the success of tissue- and cell-based therapies. Recent research has demonstrated the robust formation of human vascular networks by embedding human vascular endothelial cells and perivascular mesenchymal cells in GelMA hydrogels. Vascular cell-laden GelMA hydrogels can be microfabricated using different methodologies and integrated with microfluidic devices to generate a vasculature-on-a-chip system for disease modeling or drug screening. Bioengineered vascular networks can also serve as build-in vasculature to ensure the adequate oxygenation of thick tissue-engineered constructs. Meanwhile, several reports used GelMA hydrogels as implantable materials to deliver therapeutic cells aiming to rebuild the vasculature in ischemic wounds for repairing tissue injuries. Here, we intend to reveal present work trends and provide new insights into the development of clinically relevant applications based on vascularized GelMA hydrogels.

From Macro to Micro: Comparison of Imaging Techniques to Detect Vascular Network Formation in Left Ventricle Decellularized Extracellular Matrix Hydrogels.

Background: Angiogenesis is a crucial process in physiological maintenance and tissue regeneration. To understand the contribution of angiogenesis, it is essential to replicate this process in an environment that reproduces the biochemical and physical properties which are largely governed by the extracellular matrix (ECM). We investigated vascularization in cardiac left ventricular ECM hydrogels to mimic post-myocardial repair. We set out to assess and compare different destructive and non-destructive methods, optical as well as non-optical, to visualize angiogenesis and associated matrix remodeling in myocardial ECM hydrogels. Methods: A total of 100,000, 300,000, and 600,000 Human Pulmonary Microvascular Endothelial Cells (HPMEC) were seeded in left ventricular cardiac ECM hydrogel in 48-well plates. After 1, 7, and 14 days of culture, the HPMEC were imaged by inverted fluorescence microscopy and 3D confocal laser scanning microscopy (Zeiss Cell Discoverer 7). In addition, cell-seeded ECM hydrogels were scanned by optical coherence tomography (OCT). Fixed and paraffin-embedded gels were thin-sectioned and assessed for ECM components via H&E, picrosirius red histochemical staining, and immunostaining for collagen type I. ImageJ-based densitometry was used to quantify vascular-like networks and GraphPad was used for statistical analyses. Results: Qualitative analyses were realized through fluoromicrographs obtained by the confocal laser scanning microscope which allowed us to visualize the extensive vascular-like networks that readily appeared at all seeding densities. Quantification of networks was only possible using fluoromicrographs from inverted microscopy. These showed that, after three days, the number of master junctions was seeding density-dependent. The resolution of optical coherence tomography was too low to distinguish between signals caused by the ECM and cells or networks, yet it did show that gels, irrespective of cells, were heterogeneous. Interestingly, (immuno)histochemistry could clearly distinguish between the cast cardiac-derived matrix and newly deposited ECM in the hydrogels. The H&E staining corroborated the presence of vascular-like network structures, albeit that sectioning inevitably led to the loss of 3D structure. Conclusions: Except for OCT, all methods had complementary merit and generated qualitative and quantitative data that allowed us to understand vascular network formation in organ-derived ECM hydrogels.

Bioactivated gellan gum hydrogels affect cellular rearrangement and cell response in vascular co-culture and subcutaneous implant models

Hydrogels are suitable soft tissue mimics and capable of creating pre-vascularized tissues, that are useful for in vitro tissue engineering and in vivo regenerative medicine. The polysaccharide gellan gum (GG) offers an intriguing matrix material but requires bioactivation in order to support cell attachment and transfer of biomechanical cues. Here, four versatile modifications were investigated: Purified NaGG; avidin-modified NaGG combined with biotinylated fibronectin (NaGG-avd); oxidized GG (GGox) covalently modified with carbohydrazide-modified gelatin (gelaCDH) or adipic hydrazide-modified gelatin (gelaADH). All materials were subjected to rheological analysis to assess their viscoelastic properties, using a time sweep for gelation analysis, and subsequent amplitude sweep of the formed hydrogels. The sweeps show that NaGG and NaGG-avd are rather brittle, while gelatin-based hydrogels are more elastic. The degradation of preformed hydrogels in cell culture medium was analyzed with an amplitude sweep and show that gelatin-containing hydrogels degrade more dramatically. A co-culture of GFP-tagged HUVEC and hASC was performed to induce vascular network formation in 3D for up to 14 days. Immunofluorescence staining of the αSMA+ network showed increased cell response to gelatin-GG networks, while the NaGG-based hydrogels did not allow for the elongation of cells. Preformed, 3D hydrogels disks were implanted to subcutaneous rat skin pockets to evaluate biological in vivo response. As visible from the hematoxylin and eosin-stained tissue slices, all materials are biocompatible, however gelatin-GG hydrogels produced a stronger host response. This work indicates, that besides the biochemical cues added to the GG hydrogels, also their viscoelasticity greatly influences the biological response.

Endocannabinoids and related compounds as modulators of angiogenesis: Concepts and clinical significance.

Vasculogenesis (the process of differentiation of angioblasts toward endothelial cells and de novo formation of crude vascular networks) and angiogenesis (the process of harmonized sprouting and dispersal of new capillaries from previously existing ones) are two fundamentally complementary processes, obligatory for maintaining physiological functioning of vascular system. In clinical practice, however, the later one is of more importance as it guarantees correct embryonic nourishment, accelerates wound healing processes, prevents uncontrolled cell growth and tumorigenesis, contributes in supplying nutritional demand following occlusion of coronary vessels and is in direct relation with development of diabetic retinopathy. Hence, discovery of novel molecules capable of modulating angiogenic events are of great clinical importance. Recent studies have demonstrated multiple angio-regulatory activities for endocannabinoid system modulators and endocannabinoid-like molecules, as well as their metabolizing enzymes. Hence, in present article, we reviewed the regulatory roles of these molecules on angiogenesis and described molecular mechanisms underlying them.

The role of angiogenic factors eNOS / VEGF in the treatment of anaplastic glioma

Introduction: Anaplastic gliomas (AG) account for 6–15 % of all primary brain tumors. These include: anaplastic astrocytoma (AA), anaplastic oligodendroglioma (AO), anaplastic oligoastrocytoma (AOA), and rarer forms — anaplastic pleomorphic xanthoastrocytoma and anaplastic ganglioma. According to the data on these factors, endothelial nitric oxide synthase (eNOS) is promising in terms of the prognostic value of the course of the tumor process. It was reported that a number of vascular VEGF factors interact with eNOS, contributing to the formation of an intra-tumor vascular network, which can create conditions for uneven prolonged hypoxia, leading to the emergence of more stable tumor cells. Its role in the development of a higher anaplasia level has not been studied in isolation, which determines the relevance of this study. The prognostic role of changes in endothelial nitric oxide synthase (eNOS) in the continued growth and malignant transformation of anaplastic gliomas was studied. Results: Histological samples of brain tumors of 22 patients at the University Clinic in Nizhny Novgorod from 2017 to 2019 were examined and verified for the presence of high-grade III glioma, according to the data of the World Health Organization. The average age of the patients was 50.7 years. The material was obtained as a result of surgical removal of recurrent tumors after chemo and radiotherapy. Discussion: The microenvironment of anaplastic glioma plays an essential role in its pathogenesis. More importantly, angiogenesis, which causes the supply of glioma cells with oxygen, growth factors, nutrients, and hormones, is a significant process of tumor dissemination and growth. The degree of microvascular proliferation and angiogenesis was associated with poor survival rate, transition from a lower grade to a high grade, and relapse. In high-grade glioma, such as anaplastic glioma, neoangiogenesis is an important physiological process that provides adequate blood supply for the proliferation, survival, and invasion of glioma cells. Conclusion: The high mortality rate in gliomas underscores the urgent need for effective treatment. The glioma pathogenesis is complex and can be caused by various mechanisms, as evidenced by abnormal activation of tumor angiogenesis and mutation of isocitrate dehydrogenase. VEGF acts as a regulator of angiogenesis and is widely recognized as a critical factor in glioma development and progression. Our results suggest that VEGF and eNOS inhibition may be an effective way to control and/or block endothelial barrier damage and prevent tumor progression.

Computational Simulation of Tumor-Induced Angiogenesis.

Cancer cells require higher oxygen levels and nutrition than normal cells. Cancer cells induce angiogenesis (the development of new blood vessels) from preexisting vessels. This biological process depends on the special, chemical, and physical properties of the microenvironment surrounding tumor tissues. The complexity of these properties hinders an understanding of their mechanisms. Various mathematical models have been developed to describe quantitative relationships related to angiogenesis. We developed a three-dimensional mathematical model that incorporates angiogenesis and tumor growth. We examined angiopoietin, which regulates the spouting and branching events in angiogenesis. The simulation successfully reproduced the transient decrease in new vessels during vascular network formation. This chapter describes the protocol used to perform the simulations.

Modelling mechanically dominated vasculature development

Vascular networks play a key role in the development, function, and survival of many organisms, facilitating transport of nutrients and other critical factors within and between systems. The development of these vessel networks has been explored in a variety of in vivo, in vitro, and in silico contexts. However, the role of interactions between the growing vasculature and its environment remains largely unresolved, particularly concerning mechanical effects. Motivated by this gap in understanding, we develop a computational framework that is tailored to exploring the role of the mechanical environment on the formation of vascular networks. Here, we describe, document, implement, and explore an agent-based modelling framework, resolving the growth of individual vessels and seeking to capture phenomenology and intuitive, qualitative mechanisms. In our explorations, we demonstrate that such a model can successfully reproduce familiar network structures, whilst highlighting the roles that mechanical influences could play in vascular development. For instance, we illustrate how an external substrate could act as an effective shared memory for the periodic regrowth of vasculature. We also observe the emergence of a nuanced collective behaviour and clustered vessel growth, which results from mechanical characteristics of the external environment.

A Metabolic Signature to Monitor Endothelial Cell Differentiation, Activation, and Vascular Organization.

The formation of new blood vessels is an important step in the morphogenesis and organization of tissues and organs; hence, the success of regenerative medicine procedures is highly dependent on angiogenesis control. Despite the biotechnological advances, tissue engineering is still a challenge. Regarding vascular network formation, the regulators are well known, yet the identification of markers is pivotal in order to improve the monitoring of the differentiation and proliferation of endothelial cells, as well as the establishment of a vascular network supporting tissue viability for an efficacious implantation. The metabolic profile accompanies the physiological stages of cells involved in angiogenesis, being a fruitful hub of biomarkers, whose levels can be easily retrieved. Through NMR spectroscopy, we identified branched amino acids, acetate, and formate as central biomarkers of monocyte-to-endothelial-cell differentiation and endothelial cell proliferation. This study reinforces the successful differentiation process of monocytes into endothelial cells, allowing self-to-self transplantation of patient-derived vascular networks, which is an important step in tissue engineering, since monocytes are easily isolated and autologous transplantation reduces the immune rejection events.

Extracellular vesicles secreted by human uterine stromal cells regulate decidualization, angiogenesis, and trophoblast differentiation

In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.

MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia.

Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.

Abstract P1141: Heparinized Alginate And Collagen-based Hydrogels Enhance Localized Vascularization In Ischemic Tissue

Over 800,000 myocardial infarctions (MI) occur annually in the United States alone, 25% of which represent a repeat occurrence further indicating chronic ischemia in the myocardium. Cardiovascular regeneration is largely dependent on the stimulation of blood vessel development for recovering tissue perfusion and modulating ventricular remodeling. The delivery of angiogenic growth factors and cytokines has demonstrated the ability to initiate angiogenesis and vascular network formation in cardiac tissue, with localization of bioactive molecules more effective than systemic delivery. Alginate, with its high biocompatibility and biodegradation, represents a promising hydrogel material for factor delivery due to its high protein loading capacity. We hypothesize that covalently binding heparin to alginate will increase binding and retention to subsequently lengthen the local release duration of vascular-promoting factors. For greater tuning and biointegration, collagen and alginate hydrogel designs were optimized for both injection and co-implantation of a solid film with hiPSC-CM based ECTs in a rat model of myocardial infarction. Injectable and solid formulations showed stiffnesses of 50 +/- 2 kPa and 900 +/- 193 kPa, respectively, which was maintained for up to 6 hours of dry time. Release profiles showed sustained growth factor release, with 65% of the total content released at 7 days and 100% release in 14 days. In vitro evaluation using human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) showed cell viability and primitive network formation. In vivo subcutaneous injections showed host-vessel penetration into the delivered biomaterial within 4 days. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) loaded injections demonstrated an average lumen density of 80 +/- 15 vessels/mm 2 when compared to 4 +/- 1 vessels/mm 2 in the unloaded controls. In conclusion, we have designed a biomaterial system for disparate delivery models and shown successful vascularization of the biomaterial, demonstrating the versatile vascular regenerative capabilities of the hydrogel design.

Ifi30 Is Required for Sprouting Angiogenesis During Caudal Vein Plexus Formation in Zebrafish.

Interferon-gamma-inducible protein 30 (IFI30) is a critical enzyme that mainly exists in immune cells and functions in reducing protein disulfide bonds in endocytosis-mediated protein degradation. Regardless of this, it is also found to be expressed in vascular system. However, the functions of IFI30 in vascular development remains unknown. Vascular network formation is a tightly controlled process coordinating a series of cell behaviors, including endothelial cell (EC) sprouting, proliferation, and anastomosis. In this work, we analyzed the function of zebrafish Ifi30, orthologous to the human IFI30, in vascular development during embryogenesis. The ifi30 gene was found to be highly expressed in the caudal vein plexus (CVP) region of zebrafish embryos. Morpholino-mediated Ifi30 knockdown in zebrafish resulted in incomplete CVP formation with reduced loop numbers, area, and width. Further analyses implied that Ifi30 deficiency impaired cell behaviors of both ECs and macrophages, including cell proliferation and migration. Here, we demonstrate a novel role of IFI30, which was originally identified as a lysosomal thiol reductase involved in immune responses, in CVP development during embryogenesis. Our results suggest that Ifi30 is required for sprouting angiogenesis during CVP formation, which may offer an insight into the function of human IFI30 in angiogenesis under physiological or pathological conditions.

Smart acoustic 3D cell construct assembly with high-resolution

Precise and flexible three-dimensional (3D) cell construct assembly using external forces or fields can produce micro-scale cellular architectures with intercellular connections, which is an important prerequisite to reproducing the structures and functions of biological systems. Currently, it is also a substantial challenge in the bioengineering field. Here, we propose a smart acoustic 3D cell assembly strategy that utilizes a 3D printed module and hydrogel sheets. Digitally controlled six wave beams offer a high degree of freedom (including wave vector combination, frequency, phase, and amplitude) that enables versatile biomimetic micro cellular patterns in hydrogel sheets. Further, replaceable frames can be used to fix the acoustic-built micro-scale cellular structures in these sheets, enabling user-defined hierarchical or heterogeneous constructs through layer-by-layer assembly. This strategy can be employed to construct vasculature with different diameters and lengths, composed of human umbilical vein endothelial cells and smooth muscle cells. These constructs can also induce controllable vascular network formation. Overall, the findings of this work extend the capabilities of acoustic cell assembly into 3D space, offering advantages including innovative, flexible, and precise patterning, and displaying great potential for the manufacture of various artificial tissue structures that duplicate in vivo functions.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. β-catenin protein expression and localization were analyzed by immunocytochemistry. We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the β-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.

Surgical Approaches to Pediatric Moyamoya Disease

Moyamoya disease(MMD)is a chronic, occlusive cerebrovascular disease with unknown etiology, characterized by progressive stenosis of the internal carotid artery terminus and abnormal vascular network formation at the base of the brain. MMD has bimodal age distribution, with peaks in children and young adults, and surgical revascularization is a reasonable management choice for pediatric and adult patients with ischemic MMD. The superficial temporal artery-middle cerebral artery anastomosis with indirect pial synangiosis(combined revascularization)is a preferred surgical procedure for adult and pediatric MMD patients, providing early cerebral hemodynamic improvement and long-term favorable outcomes. Pediatric patients with MMD can present intrinsic postoperative hemodynamic complications, such as watershed shift ischemia and transient global hypoperfusion, even after successful revascularization. Therefore, perioperative management based on the early hemodynamic study is critical to avoid surgical complications, including perioperative cerebral infarction.

High-Throughput Imaging of Blood Flow Reveals Developmental Changes in Distribution Patterns of Hemodynamic Quantities in Developing Zebrafish.

Mechanical forces from blood flow and pressure (hemodynamic forces) contribute to the formation and shaping of the blood vascular network during embryonic development. Previous studies have demonstrated that hemodynamic forces regulate signaling and gene expression in endothelial cells that line the inner surface of vascular tubes, thereby modifying their cellular state and behavior. Given its important role in vascular development, we still know very little about the quantitative aspects of hemodynamics that endothelial cells experience due to the difficulty in measuring forces in vivo. In this study, we sought to determine the magnitude of wall shear stress (WSS) exerted on ECs by blood flow in different vessel types and how it evolves during development. Utilizing the zebrafish as a vertebrate model system, we have established a semi-automated high-throughput fluorescent imaging system to capture the flow of red blood cells in an entire zebrafish between 2- and 6-day post-fertilization (dpf). This system is capable of imaging up to 50 zebrafish at a time. A semi-automated analysis method was developed to calculate WSS in zebrafish trunk vessels. This was achieved by measuring red blood cell flow using particle tracking velocimetry analysis, generating a custom-made script to measure lumen diameter, and measuring local tube hematocrit levels to calculate the effective blood viscosity at each developmental stage. With this methodology, we were able to determine WSS magnitude in different vessels at different stages of embryonic and larvae growth and identified developmental changes in WSS, with absolute levels of peak WSS in all vessel types falling to levels below 0.3 Pa at 6 dpf. Additionally, we discovered that zebrafish display an anterior-to-posterior trend in WSS at each developmental stage.

Abstract 178: Multidimensional hydrogel model to benchmark progression of temozolomide-resistant glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most common and lethal form of brain cancer with a median survival of only 12 months. Due to challenges associated with surgical removal, resistance to chemo- and radiotherapy, and highly invasive nature of the tumor, GBM typically recurs rapidly near the primary tumor site, and the only widely-used therapeutic agent against GBM, temozolomide (TMZ), is merely effective. Previously, we have shown that a methacrylamide-functionalized gelatin (GelMA) hydrogel platform can support addition of brain-mimetic molecules and formation of vascular networks to study GBM behavior. In this study, we assess GBM response to TMZ using a unique set of isogenically matched GBM cell lines (8MGBA-WT vs. 8MGBA-TMZres; 42MGBA-WT vs. 42MBGA-TMZres) that exhibit TMZ responsiveness (WT) or resistance (TMZres) along with disparate proliferative and invasive patterns in two-dimensional culture. This study seeks to define changes in invasion and proliferation in response to TMZ within three-dimensional gelatin hydrogels. Methods: 8MGBA-WT, 8MGBA-TMZres, 42MGBA-WT, and 42MBGA-TMZres cell lines were cultured in DMEM with 10% FBS (100 μM TMZ continuously applied to 8MGBA-TMZres). Cells were encapsulated in GelMA hydrogels and cultured for 8 days with daily media change. After 24 hours, TMZ doses between 0 - 500 μM were applied to the gels and maintained for 7 days with daily media change. Growth rate inhibition (GR) statistics were determined after 7 days of culture. Relative cell count was assessed with the alamarBlue assay. GBM cell spheroids incapsulated in GelMA hydrogels were generated to measure relative invasion of wild type (WT) vs. TMZres lines. Spheroid outgrowth area was quantified with image analysis. Results: We observed changes in proliferative and invasive phenotypes between the cell lines consistent with 2D cell culture. Notably, 42MGBA-TMZres cells exhibited proliferative advantage, while 8MGBA-TMZres exhibited invasive advantage. GR analysis revealed apparent differences between WT and TMZres lines with the following GR50 values: 41 μM, 8MGBA-WT; 150 μM, 8MGBA-TMZres; 77 μM, 42MGBA-WT; 339 μM, 42MGBA-TMZres. These results provide insight into potential common pathways between the “go” or “grow” phenotypes that can be used as therapeutic targets for GBM. Conclusions: We have shown that a GelMA hydrogel platform can be used to assess GBM response to TMZ in 3D culture. This model provides insight into rapid tumor cell proliferation and diffusive local invasion. Further, this platform will be advanced to include a previously-developed model of GBM perivascular niche; this next-generation model will be used to benchmark the effect of perivascular niche on GBM therapeutic resistance and invasion. Hence, ongoing experiments aim to develop a vascularized hydrogel model of GBM to benchmark new TMZ variants hypothesized to overcome perivascular-induced resistance. Citation Format: Viktoriia Kriuchkovskaia, Brendan A. Harley, Rebecca B. Riggins. Multidimensional hydrogel model to benchmark progression of temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 178.

Cardiac Progenitor Cells and Adipocyte Stem Cells from Same Patients Exhibit In Vitro Functional Differences.

Cardiac progenitor cells (CPCs) and adipocyte stem cells (ASCs) are widely tested for their efficacy in repairing the diseased heart with varying results. However, no study has directly compared the functional efficacy of CPCs and ASCs collected from the same patient. CPCs and ASCs were isolated from the right atrial appendage and epicardial adipose tissue of the same patients, using explant culture. The flow cytometry analysis confirmed that both the cell types express common mesenchymal stem cells markers CD90 and CD105. ASCs, in addition, expressed CD29 and CD73. The wound-healing assay demonstrated that CPCs migrate faster to cover the wound area. Both cell types were resistant to hypoxia-induced cell death when exposed to hypoxia and serum deprivation; however, the ASCs showed increased proliferation. Conditioned medium (CM) collected after culturing serum-deprived CPCs and ASCs showed differential secretion patterns, with ASC CM showing an increased IGF-1 level, while CPC CM showed an increased FGF level. Only CPC CM reduced hypoxia-induced apoptosis in AC-16 human ventricular cardiomyocytes, while vascular network formation by endothelial cells was comparable between CPC and ASC CM. In conclusion, ASCs and CPCs exhibit differential characteristics within the same patient, and in vitro studies showed that CPCs have marginally superior functional efficacy.