Angiogenic Effect of Pravastatin Alone and with Sera from Healthy and Complicated Pregnancies Studied by in vitro Vasculogenesis/Angiogenesis Assay.

OBJECTIVE: To determine the direct effect of pravastatin on angiogenesis and to study the interaction between pravastatin and maternal sera from women with early- or late-onset pre-eclampsia (PE), intrauterine growth restriction, or healthy pregnancy. METHODS: We collected 5 maternal serum samples from each group. The effect of pravastatin on angiogenesis was assessed with and without maternal sera by quantifying tubule formation in a human-based in vitro assay. Pravastatin was added at 20, 1,000, and 8,000 ng/mL concentrations. Concentrations of angiogenic and inflammatory biomarkers in serum and in test medium after supplementation of serum alone and with pravastatin (1,000 ng/mL) were measured. RESULTS: Therapeutic concentration of pravastatin (20 ng/mL) did not have significant direct effect on angiogenesis, but the highest concentrations inhibited angiogenesis. Pravastatin did not change the levels of biomarkers in the test media. There were no changes in angiogenesis when therapeutic dose of pravastatin was added with maternal sera, but there was a trend to wide individual variation towards enhanced angiogenesis, particularly in the early-onset PE group. CONCLUSIONS: At therapeutic concentration, pravastatin alone or with maternal sera has no significant effect on angiogenesis, but at high concentrations the effect seems to be anti-angiogenic estimated by in vitro assay.

Association between [68Ga]NODAGA-RGDyK uptake and dynamics of angiogenesis in a human cell-based 3D model.

Radiolabeled RGD peptides targeting expression of αvß3 integrin have been applied to in vivo imaging of angiogenesis. However, there is a need for more information on the quantitative relationships between RGD peptide uptake and the dynamics of angiogenesis. In this study, we sought to measure the binding of [68Ga]NODAGA-RGDyK to αvß3 integrin in a human cell-based three-dimensional (3D) in vitro model of angiogenesis, and to compare the level of binding with the amount of angiogenesis. Experiments were conducted using a human cell-based 3D model of angiogenesis consisting of co-culture of human adipose stem cells (hASCs) and of human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced with four concentrations (25%, 50%, 75%, and 100%) of growth factor cocktail resulting in a gradual increase in the density of the tubule network. Cultures were incubated with [68Ga]NODAGA-RGDyK for 90 min at 37 °C, and binding of radioactivity was measured by gamma counting and digital autoradiography. The results revealed that tracer binding increased gradually with neovasculature density. In comparison with vessels induced with a growth factor concentration of 25%, the uptake of [68Ga]NODAGA-RGDyK was higher at concentrations of 75% and 100%, and correlated with the amount of neovasculature, as determined by visual evaluation of histological staining. Uptake of [68Ga]NODAGA-RGDyK closely reflected the amount of angiogenesis in an in vitro 3D model of angiogenesis. These results support further evaluation of RGD-based approaches for targeted imaging of angiogenesis.

In Vitro Vascular Network Modified to Function as Culture Platform and Angiogenic Induction Potential Test for Cancer Cells.

Drug treatments have been designed to inhibit tumor angiogenesis in hope of stopping tumor growth. However, not all tumor types respond to this type of treatment. A screening method which identifies angiogenesis inducing cancer types would help predict the efficacy of angiogenesis-inhibiting drugs for the patients. Our goal is to develop (1) a cell assay to assess the angiogenic induction potential of patient-derived tumor cells, and (2) a protocol for culturing cancer cells on a vascular platform. We optimized the media composition and seeding density of cells (hASC, HUVEC, and cancer cells) to 48-, 96-, and even 384-well plate sizes to allow vascular formation and cancer cell proliferation and subsequent analysis with high throughput. The angiogenic induction potential of patient-derived cancer cells was investigated by quantifying the formation of tubular structures and the drug response of cancer cells grown on a vascular platform was evaluated using gene expression and cell viability (WST-1) assay. Immunocytochemistry was performed with von Willebrand factor, collagen IV, CD44, cytokeratin 19 and ALDH1A1. The angiogenic induction potential test was shown to be responsive to the induction of angiogenesis by cancer cells. The responses of cancer cells were different when grown on a vascular platform or on plastic, seen in gene expression level and viability results. These two protocols are promising novel tools for aiding the selection of efficient cancer drugs for personalized medicine and as an alternative cancer cell culture platform.

Angiogenic capacity in pre-eclampsia and uncomplicated pregnancy estimated by assay of angiogenic proteins and an in vitro vasculogenesis/angiogenesis test.

OBJECTIVE: The purpose of the study was to determine the angiogenic capacity of sera in early and late pregnancy and in umbilical blood serum after childbirth, and to define how angiogenic properties assessed in a functional in vitro test are related to individual angiogenic proteins in six women with pre-eclampsia and in six healthy pregnant controls. METHODS: Maternal first and third trimester serum samples, and umbilical blood samples after childbirth, were tested in an in vitro human adipose stromal cell-human umbilical vein endothelial cell (hASC-HUVEC) vasculogenesis/angiogenesis assay. The angiogenic properties of the samples were measured by quantifying tubule formation. Concentrations of total placental growth factor (PlGF), total vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) were determined by immunoassay. RESULTS: First-trimester maternal sera of both groups had a stimulatory effect on angiogenesis in vitro and levels of angiogenic proteins did not differ between the groups. Third-trimester maternal sera in the pre-eclampsia group had an inhibitory effect on tubule formation, while those from normal pregnancies remained stimulatory. Compared with the first trimester there was a significant change in the concentrations of angiogenic proteins toward an anti-angiogenic state in pre-eclampsia. Umbilical blood serum exhibited strong anti-angiogenic effects without a significant difference between groups. CONCLUSIONS: Third-trimester serum of pre-eclamptic patients is anti-angiogenic. This phenomenon is not yet present in the first trimester. Umbilical blood serum shows inhibitory effects on angiogenesis after normal as well as pre-eclamptic pregnancy.

Differentiation of human adipose stromal cells in vitro into insulin-sensitive adipocytes.

Adipose tissue-related diseases such as obesity and type 2 diabetes are worldwide epidemics. In order to develop adipose tissue cultures in vitro that mimic more faithfully the in vivo physiology, new well-characterized and publicly accepted differentiation methods of human adipose stem cells are needed. The aims of this study are (1) to improve the existing natural adipose tissue extract (ATE)-based induction method and (2) to study the effects of a differentiation method on insulin responsiveness of the resulting adipocytes. Different induction media were applied on human adipose stromal cell (hASC) monocultures to study the differentiation capacity of the induction media and the functionality of the differentiated adipocytes. Cells were differentiated for 14 days to assess triglyceride accumulation per cell and adipocyte-specific gene expression (PPARγ, adiponectin, AP2, leptin, Glut4, Prdm16, CIDEA, PGC1-α, RIP140, UCP and ADCY5). Insulin response was studied by measuring glucose uptake and inhibition of lipolysis after incubation with 100 or 500 nM insulin. The selected differentiation method included a 3-day induction with ATE, 6 days in serum-free medium supplemented with 1.15 µM insulin and 9.06 µM Troglitazone, followed by 4 days in a defined serum- and insulin-free stimulation medium. This protocol induced prominent general adipocyte gene expression, including markers for both brown and white adipocytes and triglyceride accumulation. Moreover, the cells were sensitive to insulin as observed from increased glucose uptake and inhibition of lipolysis. This differentiation protocol provides a promising approach for the induction of hASC adipogenesis to obtain functional and mature human adipocytes.

Adipose stromal cell tubule network model provides a versatile tool for vascular research and tissue engineering.

The current limitation in designing three-dimensional tissue models is the lack of adequate vascularization with mature and stable vessels. Adipose tissue is known to secrete several angiogenic factors, and human adipose stromal cells (hASC) are known to promote vessel growth, maturation and stabilization. In this study, hASC were induced to angiogenesis with growth factor-enriched medium either in monoculture or in coculture with human umbilical vein endothelial cells (HUVEC) and analyzed for vascular, pericytic and smooth muscle cell markers. hASC and HUVEC cocultures showed an accelerated proliferation rate and the cells self-assembled, independent of the cell passage number, into multilayered three-dimensional tubular networks. The networks of hASC and HUVEC expressed endothelial markers, a complete basement membrane and vessel-supporting cells with contractile properties. A hASC and green fluorescence protein-HUVEC-infection model revealed that cocultures consisted of a mosaic of von Willebrand factor-positive cells derived from both cell populations - hASC and HUVEC. hASC monoculture had passage- and donor-dependent ability to form tubular networks, with half of the cultures presenting tubule structures and basement membrane formation. Pericytic and smooth muscle cell markers were expressed in hASC monoculture even when tubules were absent. By combining the potential properties of hASC and features from the present angiogenesis assays, we generated a natural-like, xeno-free, prevascular-like network in vitro model with excellent reproducibility and minimal limitations in technical performance. This tubular network model is an excellent tool for studying cell interactions during vascular development, for chemical and drug testing and for developing natural-like, multilayered, vascularized, scaffold-free tissue models.

Decellularized In Vitro Capillaries for Studies of Metastatic Tendency and Selection of Treatment.

Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: "network" and "cluster". Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures.

Therapeutic doses of metformin do not have impact on angiogenesis in presence of sera from pre-eclamptic, IUGR and healthy pregnancies.

Recent evidence suggests that metformin may prevent pre-eclampsia by reverting the angiogenic imbalance in maternal sera. In this study, we investigated effect of metformin on angiogenesis by quantifying tubule formation in a human-based in vitro test with co-culture of human adipose stromal cell (hASC) and human umbilical vein endothelial cell (HUVEC). A total of 20 pregnant women were recruited in the study. Serum samples were obtained from women with early- and late-onset pre-eclampsia and from women with pregnancies complicated by intrauterine growth restriction (IUGR) without pre-eclampsia (N = 5 in each of the three groups). Serum samples from women with healthy pregnancies served as controls (N = 5). The direct effect of metformin on angiogenesis was first assessed without maternal sera. Secondly, we investigated the impact of metformin on angiogenesis in the present of maternal sera. Metformin was used at 5, 50 and 600 µg/ml concentrations. Angiogenic and inflammatory biomarkers in maternal sera were analyzed by immunoassays. When the direct effect of metformin was studied, the two lowest concentrations of metformin did not affect tubule formation (angiogenesis), but the highest concentration inhibited angiogenesis. When metformin was supplemented at therapeutic concentrations of 5 and 50 µg/ml along with serum samples, there was no change in tubule formation in comparison to maternal sera alone. However, strong inhibitory effect on tubule formation was observed in all groups with the highest, non-therapeutic (600 µg/ml), concentration of metformin.

Development of novel human in vitro vascularized adipose tissue model with functional macrophages.

Inflammation has been proven significant factor in development of type 2 diabetes. So far, most of the adipose tissue related research has been performed in animals, mainly rodent models. The relevance of translation of animal results to humans is questionable. However, in vitro model with relevant human cell source, such as human adipose tissue stromal cells (hASC), can be developed and should be utilized for human adipose tissue research. We developed in vitro models of human adipose tissue utilizing hASC, endothelial cells and monocytes/macrophages. By isolating endothelial cells and macrophages from same adipose tissue as hASC, we were able to provide method for constructing personalized models of adipose tissue. With these models, we studied the effect of macrophages on adipogenesis and protein secretion, with and without vasculature. The models were analyzed for immunocytochemical markers, cell number, triglyceride accumulation and protein secretion. We found that lipid accumulation was greater in adipocytes in the presence of macrophages. Interferon gamma increased this difference between adipocyte culture and Adipocyte-Macrophage co-culture. Protein secretion was affected more by macrophages when vasculature was not present compared to the mild effect when vasculature was present. The vascularized adipose model with macrophages is valuable tool for human adipose tissue research, especially for the personalized medicine approaches; for choosing the right treatments and for studying rare medical conditions.

Presence of vasculature results in faster insulin response in adipocytes in vascularized adipose tissue model.

Besides being an energy storage, adipose tissue is an endocrine organ closely associated with vascular system. Human relevant in vitro models are needed to study adipose tissue and related diseases. Vasculature plays a central role in the development and inhibition of adipose tissue related diseases. Here, adipocyte culture was established from hASC (human adipose stromal cells), and a vascularized adipose tissue model was established from hASC and HUVEC (human umbilical cord vein endothelial cell) co-culture, utilizing the same differentiation procedure. Using these models together allowed analysis of the effect of vascularization on adipocytes. Adipocyte culture and Vascularized adipose tissue model were characterized on gene (adipocyte and vasculature-related), protein (von Willebrand factor, CollagenIV, CD140b and CD144, secretion of leptin, adiponectin and FABP4) and functional (triglyceride accumulation, glucose uptake and lipolysis) levels. Additionally, vascularized adipose tissue model was exposed to chemicals with known effects on adipogenesis and angiogenesis (rosiglitazone, chlorpyrifos, prochloraz, mancozeb, butylparaben, 15-deoxy-δ12,14-prostaglandin j2, bisphenol a, bis-(2-ethylhexyl) phthalate, tributyltin chloride) to compare their effects to the literature. The in vitro vascularized adipose tissue model showed presence of functional adipocytes and extensive vascular network. Adipocytes and the vasculature showed relevant gene and protein markers. Insulin induced glucose uptake, inhibited lipolysis and influenced vasculature-related genes. The results showed that vasculature led to faster insulin response in lipolysis inhibition and modulated responses to chemicals. This novel thoroughly characterized vascularized adipose tissue model is a promising new tool for studying adipose tissue as well as effect of chemicals on adipogenesis and angiogenesis in adipose tissue.

Development of Versatile Human In Vitro Vascularized Adipose Tissue Model with Serum-Free Angiogenesis and Natural Adipogenesis Induction.

Many adipose tissue-related diseases, such as obesity and type 2 diabetes, are worldwide epidemics. For studying these diseases, relevant human cell models are needed. In this study, we developed a vascularized adipose tissue model where human adipose stromal cells and human umbilical cord vein endothelial cells were cocultured with natural adipogenic and defined serum-free angiogenic media for 14 days. Several different protocols were compared to each other. The protocols varied in cell numbers and plating sequences. Lipid accumulation was studied with AdipoRed reagent, relative cell number with WST-1 reagent, gene expression of glut4, leptin, aP2, adiponectin, PPARγ and PPARγ2 with RT-qPCR. Secretion of adiponectin, leptin and aP2 was analysed with ELISA. The immunostained vascular network was imaged with Cell-IQ and area quantified using ImageJ. In this study, both angiogenesis and adipogenesis were successfully induced. Protocols produced strong lipid accumulation, good vascular network formation and induced adipocyte-specific protein secretion and expression of studied adipocyte genes. Results showed that cell numbers and cell plating sequences are important factors when aiming at in vitro standardized tissue model. Presence of mature vasculature appeared leads to faster the maturation of adipocytes judged by the lipid accumulation and gene expression results. The developed vascularized adipose tissue model is simple to use, easily modifiable to suit various applications and as such, a promising new tool for adipose tissue research when, for example, studying the effect of different cell types on adipose tissue function or for mechanistic studies.

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma.

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.

Cytokine-Rich Adipose Tissue Extract Production from Water-Assisted Lipoaspirate: Methodology for Clinical Use.

Proper functioning wound healing strategies are sparse. Adequate vascular formation to the injured area, as well as replacement of the volume loss, is fundamental in soft tissue repair. Tissue engineering strategies have been proposed for the treatment of these injury sites. Novel cell-free substance, human adipose tissue extract (ATE), has been previously shown to induce in vitro angiogenesis and adipogenesis and in vivo soft tissue formation. This study reports the translation of ATE preparation from laboratory to the operating room (OR). ATE samples for this study were derived from adipose tissue obtained with the water-jet assisted liposuction technique from 27 healthy patients. The variables studied included incubation time (15, 30, and 45 min), temperature (room temperature vs. 37°C), and filter type to determine the optimal method yielding the most consistent total protein content, as well as consistent and high expression of adipose-derived growth factors and cytokines, including: vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, leptin, and insulin-like growth factor. Following the optimization, samples were produced in the OR and tested for their sterility. No significant differences were observed when comparing extract incubation time points or incubation temperature. Nonetheless, when studying the different filter types used, a syringe filter with PES membrane with larger filter area showed significantly higher protein concentration (p ≤ 0.018). When studying the different growth factor concentrations, ELISA results showed less variation in cytokine concentrations in the OR samples with the optimized protocol. All of the OR samples were tested sterile. The devised protocol is an easy and reproducible OR-ready method for ATE generation. As an attractive source of growth factors, ATE is a promising alternative in the vast field of tissue engineering. Its clinical applications include volume replacement as a complement to fillers and improvement of the permanence of fat grafts and wound healing, among other bioactive functions.

Strong inhibitory effect of pre-eclampsia serum on angiogenesis detected in vitro by human cell-based angiogenesis tests.

OBJECTIVE: To explore in vitro angiogenic properties of maternal and umbilical cord blood sera from women with symptomatic pre-eclampsia in comparison with sera from women with normotensive pregnancies. STUDY DESIGN: Maternal and umbilical blood serum samples were collected from eleven primiparous women with pre-eclampsia and ten healthy gestational-age-matched primiparous controls. The samples were tested for tubule formation in two different types of in vitro angiogenesis tests. The first test (fibroblast-HUVEC) showed effects on angiogenesis and the second test (hASC-HUVEC), in addition to angiogenesis, also showed effects on vasculogenesis. The pro-angiogenic and inhibitory properties of the samples were microscopically quantified after immunostaining tubular structures, using markers for von Willebrand factor (vWf) and collagen IV. RESULTS: Serum samples from pre-eclamptic women inhibited tubule formation in both models, while those from normal pregnancy didn't. Umbilical blood samples were inhibitory both after pre-eclampsia and normal pregnancy. In the fibroblast-HUVEC model the inhibition was stronger after preeclampsia pregnancy, and the difference between groups was statistically significant. In the pre-eclampsia group a correlation between the inhibitory effect of umbilical blood and birth weight adjusted to gestational age was found. No clear correlation between sera from pregnant women and corresponding umbilical sera was found. CONCLUSION: The strong inhibitory effect of maternal serum samples on tubule formation reflects the anti-angiogenic state that is present in pre-eclampsia.

Human vascular model with defined stimulation medium - a characterization study.

The formation of blood vessels is a vital process in embryonic development and in normal physiology. Current vascular modelling is mainly based on animal biology leading to species-to-species variation when extrapolating the results to humans. Although there are a few human cell based vascular models available these assays are insufficiently characterized in terms of culture conditions and developmental stage of vascular structures. Therefore, well characterized vascular models with human relevance are needed for basic research, embryotoxicity testing, development of therapeutic strategies and for tissue engineering. We have previously shown that the in vitro vascular model based on co-culture of human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) is able to induce an extensive vascular-like network with high reproducibility. In this work we developed a defined serum-free vascular stimulation medium (VSM) and performed further characterization in terms of cell identity, maturation and structure to obtain a thoroughly characterized in vitro vascular model to replace or reduce corresponding animal experiments. The results showed that the novel vascular stimulation medium induced intact and evenly distributed vascular-like network with morphology of mature vessels. Electron microscopic analysis assured the three-dimensional microstructure of the network containing lumen. Additionally, elevated expressions of the main human angiogenesis-related genes were detected. In conclusion, with the new defined medium the vascular model can be utilized as a characterized test system for chemical testing as well as in creating vascularized tissue models.

Novel in vitro cardiovascular constructs composed of vascular-like networks and cardiomyocytes.

The interaction between different cardiac cells has shown to be important for critical biological properties including cell survival, proliferation, differentiation and function. The improvement of culture conditions with different cell types and to study their effects on cardiomyocyte viability and functionality is essential. For practical applications including general toxicity testing, drug development and tissue engineering it is important to study whether co-cultures have additional advantages over cardiomyocyte monoculture. Two multicellular in vitro cardiovascular constructs devoid of added biomaterial were developed in this study. In the first construct, neonatal rat cardiomyocytes (CM) were seeded on vascular-like network formed by human umbilical vein endothelial cells (HUVEC) and human adipose stromal cells (hASC). In the second construct, CMs were seeded on vascular-like network formed by HUVECs and human foreskin fibroblasts. The ability of these two vascular-like networks to support the viability and functionality of CMs was analyzed. Different culture media compositions were evaluated to support the development of optimal cardiovascular construct. Our results demonstrate that both vascular-like networks markedly improved CM viability and functionality. In the constructs, co-localization of CMs and vascular-like networks was seen. Multicellular constructs also allowed synchronized contractility of CMs. Serum-free medium supplemented with vascular endothelial growth factor and basic fibroblast growth factor was found to provide the most optimal conditions for cardiovascular construct as an entity. In conclusion, when combining a vascular-like network with CMs, the viability and functionality of CMs was markedly improved. The results suggest that the cardiovascular constructs developed provide a promising new tool for the assessment of toxicological and safety pharmacological effects of compounds in vitro.