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Telocytes (TCs), a novel type of mesenchymal or interstitial cell with specific, very long and thin cellular prolongations, have been found in various mammalian organs and have potential biological functions. However, their existence during lung development is poorly understood. This study aimed to investigate the existence, morphological features, and role of CD34+ SCs/TCs in mouse lungs from foetal to postnatal life using primary cell culture, double immunofluorescence, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The immunofluorescence double staining profiles revealed positive expression of CD34 and PDGFR-α, Sca-1 or VEGFR-3, and the expression of these markers differed among the age groups during lung development. Intriguingly, in the E18.5 stage of development, along with the CD34+ SCs/TCs, haematopoietic stem cells and angiogenic factors were also significantly increased in number compared with those in the E14.5, E16.5, P0 and P7. Subsequently, TEM confirmed that CD34+ SCs/TCs consisted of a small cell body with long telopodes (Tps) that projected from the cytoplasm. Tps consisted of alternating thin and thick segments known as podomers and podoms. TCs contain abundant endoplasmic reticulum, mitochondria and secretory vesicles and establish close connections with neighbouring cells. Furthermore, SEM revealed characteristic features, including triangular, oval, spherical, or fusiform cell bodies with extensive cellular prolongations, depending on the number of Tps. Our findings provide evidence for the existence of CD34+ SCs/TCs, which contribute to vasculogenesis, the formation of the airâblood barrier, tissue organization during lung development and homoeostasis.
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BACKGROUND: Prediabetes (PD) is associated with intermediate hyperglycaemia, dyslipidaemia, reduced nitric oxide (NO) bioavailability and moderate hypertension. All these factors are risk factor for preeclampsia (PE). However, the effects of the PD on placental function have not been shown. Accordingly, this study sought to investigate a possible link between maternal PD and the risk of developing PE. METHODS: Pregnant female Sprague-Dawley rats (N = 18) were divided into normal, preeclamptic and prediabetic groups (n = 6 in each group) to study the effects of maternal PD on placenta function over the period of 19 days. Blood glucose and blood pressure were measured on gestational day (GND) 0, 9 and 18. Placental vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1) mRNA expression were measured terminally. Data were analysed using ANOVA followed by the Tukey-Kramer post hoc test. Values of p < .05 were used to indicate statistical significance. RESULTS: Maternal PD and PE significantly increased blood glucose, decrease NO concentration and increase in MAP by comparison to the normal pregnant control group. Maternal PD significantly decreased VEGF, PlGF mRNA expression with a slight increase in sFlt-1 mRNA expression comparison to the normal pregnant control group. CONCLUSIONS: Maternal PD is associated with placental dysfunction due to impaired glucose handling, endothelial dysfunction and an imbalance in angiogenic and antiangiogenic factors. Therefore, maternal PD is a risk factor of PE.
People with prediabetes (PD) are at risk of developing type 2 diabetes. Studies have shown that PD can cause blood vessel problems in both men and women. However, there have not been any studies on prediabetic pregnant women, so we do not know much about the pregnancy problems they might face. Looking into new factors related to blood vessel growth and health in PD could help us understand how to diagnose and manage PD during pregnancy. This could reduce the risk of problems similar to pre-eclampsia. Research in this area will help mothers and their doctors be more aware of the complications PD can cause during pregnancy. This could lead to fewer health problems and deaths for both mothers and babies linked to type 2 diabetes.
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Glucemia , Factor de Crecimiento Placentario , Placenta , Preeclampsia , Estado Prediabético , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Femenino , Animales , Embarazo , Preeclampsia/fisiopatología , Preeclampsia/etiología , Estado Prediabético/complicaciones , Estado Prediabético/fisiopatología , Factor de Crecimiento Placentario/sangre , Ratas , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/metabolismo , Placenta/metabolismo , Factores de Riesgo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Glucemia/análisis , Glucemia/metabolismo , Presión Sanguínea , Óxido Nítrico/metabolismo , Óxido Nítrico/sangre , Modelos Animales de EnfermedadRESUMEN
Introduction: Autologous mononuclear cells (MNCs) have been used in vascular regenerative therapy since the identification of endothelial progenitor cells (EPCs). However, the efficacy of autologous EPC therapy for diseases such as diabetes and connective tissue disorders is limited due to deficiencies in the number and function of EPCs. To address this, we developed a novel RE-01 cells that enriches pro-angiogenic cells from peripheral blood MNCs (PBMNCs). Methods: PBMNCs were collected from healthy volunteers following ethical guidelines. RE-01 cells were cultured in the presence of specific growth factors for 5 days without media change. Flow cytometry was used to analyze cell surface markers. Tube formation assays, EPC culture assays, and mRNA analysis were performed to evaluate angiogenic potential. The efficacy of RE-01 cells upon transplantation into ischemic hind limbs of mice was evaluated. Results: RE-01 cells exhibited a significant increase in pro-angiogenic cells such as M2 macrophages and angiogenic T cells, in contrast to PBMNCs, while the number of inflammatory cells reduced. In vitro assays demonstrated the enhanced angiogenic abilities of RE-01 cells, supported by increased mRNA expression of angiogenesis-related cytokines. In vivo studies using mouse ischemic hind limb models have shown that blood flow and angiogenesis improved following RE-01 cell transplantation. Transplantations for 3 consecutive days significantly improved the number of pericyte-recruited vessels in the severely ischemic hind limbs of mice. Conclusions: RE-01 cells showed promising results in enhancing angiogenesis and arteriogenesis, possibly owing to the presence of M2 macrophages and angiogenic T cells. These cells also demonstrated anti-fibrotic effects. The efficacy of RE-01 cells has been confirmed in mouse models, suggesting their potential for treating ischemic vascular diseases. Clinical trials are planned to validate the safety and efficacy of RE-01 cell therapy in patients with connective tissue disease and unhealed ulcers. We hope that this new RE-01 cell therapy will prevent many patients from undergoing amputation.
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The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.
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Polycyclic aromatic hydrocarbons (PAHs) widely present in the environment, but their effect on cerebrovascular development has been rarely reported. In this study, dechorionated zebrafish embryos at 24 hpf were exposed to benzo(a)pyrene (BaP) at 0.5, 5 and 50 nM for 48 h, cerebrovascular density showed a significant reduction in the 5 and 50 nM groups. The expression of aryl hydrocarbon receptor (AhR) was significantly increased. Transcriptomic analysis showed that the pathway of positive regulation of vascular development was down-regulated and the pathway of inflammation response was up-regulated. The transcription of main genes related to vascular development, such as vegf, bmper, cdh5, f3b, itgb1 and prkd1, was down-regulated. Addition of AhR-specific inhibitor CH233191 in the 50 nM BaP group rescued cerebrovascular developmental defects and down-regulation of relative genes, suggesting that BaP-induced cerebrovascular defects was AhR-dependent. The cerebrovascular defects were persistent into adult fish raised in clean water, showing that the relative area of vascular network, the length of vessels per unit area and the number of vascular junctions per unit area were significantly decreased in the 50 nM group. Supplementation of berberine (BBR), a naturally derived medicine from a Chinese medicinal herb, alleviated BaP-induced cerebrovascular defects, accompanied by the restoration of altered expression of AhR and relative genes, which might be due to that BBR promoted BaP elimination via enhancing detoxification enzyme activities, suggesting that BBR could be a potential agent in the prevention of cerebrovascular developmental defects caused by PAHs.
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Benzo(a)pireno , Berberina , Contaminantes Químicos del Agua , Pez Cebra , Animales , Benzo(a)pireno/toxicidad , Contaminantes Químicos del Agua/toxicidad , Berberina/farmacología , Embrión no Mamífero/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidadRESUMEN
Endothelial cells (ECs) migration is a crucial early step in vascular repair and tissue neovascularization. While extensive research has elucidated the biochemical drivers of endothelial motility, the impact of biophysical cues, including vessel geometry and topography, remains unclear. Herein, we present a novel approach to reconstruct 3D self-assembly blood vessels-on-a-chip that accurately replicates real vessel geometry and topography, surpassing conventional 2D flat tube formation models. This vessels-on-a-chip system enables real-time monitoring of vasculogenesis and ECs migration at high spatiotemporal resolution. Our findings reveal that ECs exhibit increased migration speed and directionality in response to narrower vessel geometries, transitioning from a rounded to a polarized morphology. These observations underscore the critical influence of vessel size in regulating ECs migration and morphology. Overall, our study highlights the importance of biophysical factors in shaping ECs behavior, emphasizing the need to consider such factors in future studies of endothelial function and vessel biology.
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Vasos Sanguíneos , Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Células Endoteliales/citología , Dispositivos Laboratorio en un Chip , Neovascularización FisiológicaRESUMEN
Xenohormesis proposes that phytochemicals produced to combat stressors in the host plant exert biochemical effects in animal cells lacking cognate receptors. Xenohormetic phytochemicals such as flavonoids and phytoalexins modulate a range of human cell signaling mechanisms but functional correlations with human pathophysiology are lacking. Here, potent inhibitory effects of grapefruit-derived Naringenin (Nar) and soybean-derived Glyceollins (Gly) in human microphysiological models of bulk tissue vasculogenesis and tumor angiogenesis are reported. Despite this interference of vascular morphogenesis, Nar and Gly are not cytotoxic to endothelial cells and do not prevent cell cycle entry. The anti-vasculogenic effects of Glyceollin are significantly more potent in sex-matched female (XX) models. Nar and Gly do not decrease viability or expression of proangiogenic genes in triple negative breast cancer (TNBC) cell spheroids, suggesting that inhibition of sprouting angiogenesis by Nar and Gly in a MPS model of the (TNBC) microenvironment are mediated via direct effects in endothelial cells. The study supports further research of Naringenin and Glyceollin as health-promoting agents with special attention to mechanisms of action in vascular endothelial cells and the role of biological sex, which can improve the understanding of dietary nutrition and the pharmacology of phytochemical preparations.
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Flavanonas , Neovascularización Patológica , Fitoquímicos , Neoplasias de la Mama Triple Negativas , Humanos , Flavanonas/farmacología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Fitoquímicos/farmacología , Femenino , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Pterocarpanos/farmacología , Inhibidores de la Angiogénesis/farmacología , Línea Celular Tumoral , Glycine max/química , Citrus paradisi/química , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , AngiogénesisRESUMEN
Stem cell research is the product of cumulative, integrated effort between and within laboratories and disciplines. The many collaborative steps that lead to that special "Eureka moment", when something that has been a puzzle perhaps for years suddenly become clear, is among the greatest pleasures of a scientific career. In this essay, the serendipitous pathway from first acquaintance with pluripotent stem cells to advanced cardiovascular models that emerged from studying development and disease will be described. Perhaps inspiration for later generations of stem cell researchers simply to follow whatever they find interesting.
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INTRODUCTION: Heparan 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. METHODS: Human-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. RESULTS: HS 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. CONCLUSIONS: Exogenous 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.
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Diferenciación Celular , Pulpa Dental , Dentinogénesis , Heparitina Sulfato , Células Madre , Pulpa Dental/citología , Pulpa Dental/irrigación sanguínea , Humanos , Dentinogénesis/efectos de los fármacos , Dentinogénesis/fisiología , Células Madre/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Animales , Ratas , Células Cultivadas , Neovascularización Fisiológica/efectos de los fármacos , Odontogénesis/efectos de los fármacosRESUMEN
Cancer vasculogenesis is a pivotal focus of cancer research and treatment given its critical role in tumor development, metastasis, and the formation of vasculogenic microenvironments. Traditional approaches to investigating cancer vasculogenesis face significant challenges in accurately modeling intricate microenvironments. Recent advancements in three-dimensional (3D) bioprinting technology present promising solutions to these challenges. This review provides an overview of cancer vasculogenesis and underscores the importance of precise modeling. It juxtaposes traditional techniques with 3D bioprinting technologies, elucidating the advantages of the latter in developing cancer vasculogenesis models. Furthermore, it explores applications in pathological investigations, preclinical medication screening for personalized treatment and cancer diagnostics, and envisages future prospects for 3D bioprinted cancer vasculogenesis models. Despite notable advancements, current 3D bioprinting techniques for cancer vasculogenesis modeling have several limitations. Nonetheless, by overcoming these challenges and with technological advances, 3D bioprinting exhibits immense potential for revolutionizing the understanding of cancer vasculogenesis and augmenting treatment modalities.
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The secreted factor Epidermal growth factor-like protein 7 (EGFL7) is involved in angiogenesis, vasculogenesis, as well as neurogenesis. Importantly, EGFL7 is also implicated in various pathological conditions, including tumor angiogenesis in human cancers. Thus, understanding the mechanisms through which EGFL7 regulates and promotes blood vessel formation is of clear practical importance. One principle means by which EGFL7's function is investigated is via the expression and purification of the recombinant protein. This mini-review describes three methods used to produce recombinant EGFL7 protein. First, a brief overview of EGFL7's genetics, structure, and function is provided. This is followed by an examination of the advantages and disadvantages of three common expression systems used in the production of recombinant EGFL7; (i) Escherichia coli (E. coli), (ii) human embryonic kidney (HEK) 293 cells or other mammalian cells, and (iii) a baculovirus-based Sf9 insect cell expression system. Based on the available evidence, we conclude that the baculovirus-based Sf9 insect cell expression currently has the advantages of producing active recombinant EGFL7 in the native conformation with the presence of acceptable posttranslational modifications, while providing sufficient yield and stability for experimental purposes.
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Background Human embryo vasculogenesis (blood vessel development starting from endothelial precursors) includes the ability of mesenchymal cells and pluripotent stem cells to differentiate into endothelial cells. Quantification of endothelial progenitor cells is difficult to assess during the early steps of human embryo development due to several factors, especially due to the paucity of human embryo tissue which is usually discarded after early-stage pregnancy abortive methods. CD133 (Promimin-1) is a general marker of progenitor cells, but combined with other endothelial markers such as CD34, it may identify endothelial progenitor cells during embryonic development. CD34 immunohistochemistry was previously performed by our team to identify human embryo capillaries and comparatively assess microvessel density between different human embryonic tissues. TIE2 is an angiopoietin receptor strongly involved in the newly formed blood vessel maturation due to its expression in some mesenchymal precursors for future pericytes. CD34 assesses the presence of endothelial cells but its single use does not evaluate the endothelial progenitor state as CD133 may do nor vessel maturation as TIE2 may do. Data about the dynamics of CD133/TIE2 expression in the early stages of human embryo development are scarce. Hence, in this study, we aimed to comparatively assess the dynamic of CD133+ endothelial precursors and TIE2 expression on five and seven-week-old human embryonic tissues with a special emphasis on their expression on embryonic vascular beds. Methodology CD133 and TIE2 immunohistochemistry was performed on five and seven-week-old human embryonic tissues followed by their quantification using the Qu Path digital image analysis (DIA) automated method. Results CD133 and TIE2 showed divergent patterns of expression during the initial phases of human embryonic development, specifically in the vascular endothelium of tiny capillaries. The expression of CD133 in endothelial cells lining the perfused lumen gradually decreased from five to seven-week-old embryos. It remained expressed with greater intensity in cells located at the tip of the vascular bud that emerged into pre-existing capillaries. TIE2 was much more specific than CD133, being restricted to the level of the vascular endothelium; therefore, it was easier to quantify using digital image analysis. The endothelium of the embryonic aorta was an exception to the divergent expression, as CD133 and TIE2 were consistently co-expressed in the seven-week-old embryo. The Qu Path DIA assessment increased the accuracy of CD133 and TIE2 evaluation, being the first time they were quantified by using automated software and not manually. Conclusions High heterogeneity of CD133 and TIE2 was observed between five and seven-week-old embryonic tissues as well as between different embryonic regions from the same gestational age. The unique finding of CD133/TIE2 co-expression persistence inside aortic endothelium needs further studies to elucidate the role of this co-expression.
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Vasculogenesis, which refers to the development of blood vessels from precursor cells, is a process that occurs predominantly during early embryonic life. It plays a crucial role in the establishment of the primitive vascular network. Vasculogenesis diminishes throughout the fetal vascular remodeling process, giving way to angiogenesis, which becomes the predominant mechanism after birth. At first, the development of the kidney's blood vessels depends on vasculogenesis, and then both vasculogenesis and angiogenesis happen simultaneously. Both processes are necessary for the normal development of the renal vasculature. Although the kidneys are highly vascularized, our understanding of normal kidney vasculogenesis is still incomplete. This lack of knowledge may explain the limited data available on the role of vasculogenesis in the progression and spread of renal cancers. In other types of cancer, researchers have well documented the phenomenon of tumor vasculogenesis. However, there is currently limited and fragmented information about the occurrence of clear-cell renal cell carcinomas (cc-RCC). In this article, we provide a comprehensive review of the current understanding of normal kidney vasculogenesis and vasculogenic pathways in clear cell renal cell carcinoma (cc-RCC). We specifically focus on cellular precursors, growth factors, and the influence of the normal and tumor environments on these processes. It will carefully look at how tumor vasculogenesis might affect the growth and metastasis of clear cell renal cell carcinoma (cc-RCC), as well as how it might affect the effectiveness of drugs and the development of therapy resistance.
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Bone marrow-derived cells (BMDCs) are heterogeneous populations in which not only pluripotent stem cells, namely, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSC) but also endothelial progenitor cells (EPC) are involved. BMDCs contribute to the maintenance of homeostasis and recovery from disrupted homeostasis as the immune, endocrine, and nervous systems. The skin is the largest organ in which various tissues, such as the epidermis, dermis, skin appendages (i.e., hair follicles), fats, muscles, and vessels, are tightly and systematically packed. It functions as a physical barrier to block the invasion of harmful substances and pathogenic microorganisms and properly regulate water evaporation. The skin is exposed to injuries from external stimuli because it is the outermost layer and owing to its specificity. Recovery from physical injuries and DNA mutations occurs constantly in the skin, but medical treatments are required for impaired wound healing. Recently, conservative treatments utilizing scaffolds have attracted attention as alternatives to surgical therapy, which is highly invasive. Against this background, numerous scaffolds are available in a clinical setting, although they have not surpassed surgery because of their distinct disadvantages. Here, we discuss the plasticity of BMDCs in the skin to maintain homeostasis, in addition to their critical roles on recovery from disrupted homeostasis. We also share our perspective on how scaffolds can be developed to establish scaffolds beyond surgery to regenerate skin structure during wound healing by maximally utilizing the plasticity of BMDCs.
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Kidney cancers comprise about 3% of all new malignancies in the United States. Renal cell carcinomas (RCCs) are the most common type of renal malignancy making up about 85% of kidney cancer cases. Signs and symptoms of renal cell carcinomas can result from local tumor growth, paraneoplastic syndromes, or distant metastases. The classic triad of presentation with flank pain, hematuria, and a palpable abdominal mass occurs in fewer than 10% of patients. Most diagnoses result from incidental imaging findings (ultrasonography or abdominal CT imaging) performed for another reason. Localized disease is treated by partial nephrectomy, total nephrectomy, or ablation (tumor destruction with heat or cold). When the tumors have metastasized, systemic therapy with protein-tyrosine kinase antagonists including sorafenib, sunitinib, pazopanib, and tivozanib that target vascular endothelial, platelet-derived, fibroblast, hepatocyte, and stem cell factor growth factor receptors (VEGFR, PDGFR, FGFR, MET, and Kit) were prescribed after 2005. The monoclonal antibody immune checkpoint inhibitor nivolumab (targeting programed cell death protein 1, PD1) was approved for the treatment of RCCs in 2015. It is usually used now in combination with ipilimumab (targeting CTLA-4) or cabozantinib (a multikinase blocker). Other combination therapies include pembrolizumab (targeting PD1) and axitinib (a VEGFR and PDGFR blocker) or lenvatinib (a multikinase inhibitor). Since the KEYNOTE-426 clinical trial, the use of immune checkpoint inhibitors in combination with protein-tyrosine kinase inhibitors is now the standard of care for most patients with metastatic renal cell carcinomas and monotherapies are used only in those individuals who cannot receive or tolerate immune checkpoint inhibitors.
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Carcinoma de Células Renales , Inhibidores de Puntos de Control Inmunológico , Neoplasias Renales , Inhibidores de Proteínas Quinasas , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
A thiolated RGD was incorporated into the threaded allyl-ß-cyclodextrins (Allyl-ß-CDs) of the polyrotaxane (PR) through a thiol-ene click reaction, resulting in the formation of dynamic RGD ligands on the PR surface (dRGD-PR). When maintaining consistent RGD density and other physical properties, endothelial cells (ECs) cultured on dRGD-PR exhibited significantly increased cell proliferation and a larger cell spreading area compared to those on the non-dynamic RGD (nRGD-PCL). Furthermore, ECs on dRGD-PR demonstrated elevated expression levels of FAK, p-FAK, and p-AKT, along with a larger population of cells in the G2/M stage during cell cycle analysis, in contrast to cells on nRGD-PCL. These findings suggest that the movement of the RGD ligands may exert additional beneficial effects in promoting EC spreading and proliferation, beyond their essential adhesion and proliferation-promoting capabilities, possibly mediated by the RGD-integrin-FAK-AKT pathway. Moreover, in vitro vasculogenesis tests were conducted using two methods, revealing that ECs cultured on dRGD-PR exhibited much better vasculogenesis than nRGD-PCL in vitro. In vivo testing further demonstrated an increased presence of CD31-positive tissues on dRGD-PR. In conclusion, the enhanced EC spreading and proliferation resulting from the dynamic RGD ligands may contribute to improved in vitro vasculogenesis and in vivo vascularization.
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Proliferación Celular , Ciclodextrinas , Oligopéptidos , Humanos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclodextrinas/química , Ciclodextrinas/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Ligandos , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/farmacología , Oligopéptidos/química , Poloxámero/química , Poloxámero/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , RotaxanosRESUMEN
Recent advances in regenerative medicine and tissue engineering have enabled the biofabrication of three-dimensional (3D) tissue analogues with the potential for use in transplants and disease modeling. However, the practical use of these biomimetic tissues has been hindered by the challenge posed by reconstructing anatomical-scale micro-vasculature tissues. In this study, we suggest that co-cultured spheroids within hydrogels hold promise for regenerating highly vascularized and innervated tissues, bothin vitroandin vivo. Human adipose-derived stem cells (hADSCs) and human umbilical vein cells (HUVECs) were prepared as spheroids, which were encapsulated in gelatin methacryloyl hydrogels to fabricate a 3D pre-vascularized tissue. The vasculogenic responses, extracellular matrix production, and remodeling depending on parameters like co-culture ratio, hydrogel strength, and pre-vascularization time forin vivointegration with native vessels were then delicately characterized. The co-cultured spheroids with 3:1 ratio (hADSCs/HUVECs) within the hydrogel and with a pliable storage modulus showed the greatest vasculogenic potential, and ultimately formedin vitroarteriole-scale vasculature with a longitudinal lumen structure and a complex vascular network after long-term culturing. Importantly, the pre-vascularized tissue also showed anastomotic vascular integration with host blood vessels after transplantation, and successful vascularization that was positive for both CD31 and alpha-smooth muscle actin covering 18.6 ± 3.6µm2of the luminal area. The described co-cultured spheroids-laden hydrogel can therefore serve as effective platform for engineering 3D vascularized complex tissues.
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Hidrogeles , Ingeniería de Tejidos , Humanos , Ingeniería de Tejidos/métodos , Hidrogeles/química , Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana , Medicina Regenerativa , Andamios del Tejido/químicaRESUMEN
Hydrogel-based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo. However, due to their complex multifactorial regulation, adapting these tissue and disease models for high-throughput screening workflows remains challenging. In this study, a new precision culture scaling (PCS-X) methodology combines statistical techniques (design of experiment and multiple linear regression) with automated, parallelized experiments and analyses to customize hydrogel-based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation, and media composition are systematically explored to induce vasculogenesis in endothelial mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384-well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound- and dose-dependent manner, demonstrating the scope and power of PCS-X in creating parallelized tissue and disease models for drug discovery and individualized therapies.
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Células Endoteliales de la Vena Umbilical Humana , Neovascularización Fisiológica , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Hidrogeles/química , Técnicas de Cocultivo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Pericitos/citología , Pericitos/metabolismo , Pericitos/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/instrumentación , Células Endoteliales/citología , Células Endoteliales/metabolismoRESUMEN
We propose a continuum model for pattern formation, based on the multiphase model framework, to explore in vitro cell patterning within an extracellular matrix (ECM). We demonstrate that, within this framework, chemotaxis-driven cell migration can lead to the formation of cell clusters and vascular-like structures in 1D and 2D respectively. The influence on pattern formation of additional mechanisms commonly included in multiphase tissue models, including cell-matrix traction, contact inhibition, and cell-cell aggregation, are also investigated. Using sensitivity analysis, the relative impact of each model parameter on the simulation outcomes is assessed to identify the key parameters involved. Chemoattractant-matrix binding is further included, motivated by previous experimental studies, and found to reduce the spatial scale of patterning to within a biologically plausible range for capillary structures. Key findings from the in-depth parameter analysis of the 1D models, both with and without chemoattractant-matrix binding, are demonstrated to translate well to the 2D model, obtaining vascular-like cell patterning for multiple parameter regimes. Overall, we demonstrate a biologically-motivated multiphase model capable of generating long-term pattern formation on a biologically plausible spatial scale both in 1D and 2D, with applications for modelling in vitro vascular network formation.
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Quimiotaxis , Matriz Extracelular , Modelos Biológicos , Quimiotaxis/fisiología , Matriz Extracelular/fisiología , Matriz Extracelular/metabolismo , Humanos , Movimiento Celular/fisiología , Simulación por ComputadorRESUMEN
The global population is aging rapidly, posing unprecedented challenges to health care systems. This study investigates the often-overlooked role of macrophages in microvascular dysfunction associated with aging. We use a three-dimensional in vitro hydrogel model to assess the effects of both age and metformin, an anti-aging therapeutic, on macrophage interactions with microvasculature. Metformin's broad cellular impact is a subject of significant interest, yet its precise mechanisms remain unclear. Our research reveals that metformin treatment enhances genetic pathways associated with macrophage-mediated support of angiogenesis, resulting in increased microvessel density. Of importance, monocyte chemoattractant protein-1 expression is upregulated with metformin treatment and positively correlated with microvascular volume, shedding light on a potential mechanism for metformin's promotion of macrophage support of vasculogenesis. This work not only uncovers metformin's impact on human macrophages but also supports its potential as an antiaging therapeutic, offering new avenues for combating age-related diseases.