Dietary Nucleotides Retard Oxidative Stress-Induced Senescence of Human Umbilical Vein Endothelial Cells.

Several lines of evidence suggest an inhibitory role of dietary nucleotides (NTs) against oxidative stress and inflammation, which promote senescence in age-associated cardiovascular diseases. We sought to test whether the dietary NTs could retard the hydrogen peroxide (H2O2)-induced senescence of human umbilical vein endothelial cells (HUVECs) and to elucidate the efficiency of different NTs as well as the potential mechanism. Senescence was induced in HUVECs by 4 h exposure to 200 µM H2O2 and was confirmed using senescence-associated-ß-galactosidase staining (SA-ß-gal), cell viability, and Western blot analyses of p16INK4A and p21Waf1/Cip1 after 24 h administration of growth medium. We find that NTs retards oxidative stress-induced HUVECs senescence, as shown by a lower percentage of SA-ß-gal-positive cells, lower expression of p16INK4A, and p21Waf1/Cip1 as well as higher cell viability. GMP100 was the most excellent in delaying HUVECs senescence, which was followed by the NTs mixture, NMN, CMP50, and UMP50/100, while AMP retards HUVECs senescence by specifically reducing p15INK4b expression. NTs all have significant anti-inflammatory effects; AMP and CMP were more prominent in restoring mitochondrial function, GMP and CMP were more competent at eliminating ROS and MDA, while AMP and UMP were more efficient at enhancing antioxidant enzyme activity. The role of the NTs mixture in retarding HUVECs senescence is full-scaled. These results stated that the mechanisms of NTs retarding HUVECs senescence could be related to its antioxidant and anti-inflammation properties promoting cell proliferation and protecting mitochondrial function activities.

Human urinary kininogenase reduces the endothelial injury by inhibiting Pyk2/MCU pathway.

The injury of endothelial cells is one of the initiating factors in restenosis after endovascular treatment. Human urinary kallidinogenase (HUK) is a tissue kallikrein which is used for ischemia-reperfusion injury treatment. Studies have shown that HUK may be a potential therapeutic agent to prevent stenosis after vascular injury, however, the precise mechanisms have not been fully established. This study is to investigate whether HUK can protect endothelial cells after balloon injury or H2O2-induced endothelial cell damage through the proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway. Intimal hyperplasia, a decrease of pinocytotic vesicles and cell apoptosis were found in the common carotid artery balloon injury and H2O2-induced endothelial cell damage, Pyk2/MCU was also up-regulated in such pathological process. HUK could prevent these injuries partially via the bradykinin B2 receptor by inhibiting Pyk2/MCU pathway, which prevented the mitochondrial damage, maintained calcium balance, and eventually inhibited cell apoptosis. Furthermore, MCU expression was not markedly increased if Pyk2 was suppressed by shRNA technique in the H2O2 treatment group, and cell viability was significantly better than H2O2-treated only. In short, our results indicate that the Pyk2/MCU pathway is involved in endothelial injury induced by balloon injury or H2O2-induced endothelial cell damage. HUK plays an protective role by inhibiting the Pyk2/MCU pathway in the endothelial injury.

Endothelial Recovery in Bare Metal Stents and Drug-Eluting Stents on a Single-Cell Level.

OBJECTIVE: Healing processes, particularly reendothelialization, are essential for vascular homeostasis after plain old balloon angioplasty and stent implantation. Drug-eluting stents (DES) are commonly used for percutaneous coronary intervention because restenosis rates are reduced as compared with bare metal stents (BMS). However, in addition to understanding the nature of regenerated endothelial cells, concerns over incomplete stent healing persist, and the molecular effects of antiproliferative drug coatings on endothelium remain poorly understood. APPROACH AND RESULTS: We used the rabbit iliac artery model to analyze differences in stent endothelialization in BMS and DES. Histology and immunohistochemistry confirmed that stent coverage was significantly greater in BMS than in DES at 30 days after stent implantation. Single-cell RNA sequencing revealed a more immature transcriptomic signature of neointimal endothelial cell harvested from stented arteries in comparison with native and plain old balloon angioplasty­ treated arteries. Whereas the genetic signature of BMS was overall proangiogenic with enrichment of genes involved in endothelial proliferation, sprouting, and migration, as well as extracellular matrix assembly, DES-derived endothelial cell showed upregulation of genes associated with angiogenesis inhibition and endothelial activation. CONCLUSIONS: Single-cell RNA sequencing analysis identified unique transcriptional changes within regenerated endothelium after plain old balloon angioplasty and stent implantation. These data suggest unique endothelial transcriptional differences, which characterize the different response of the endothelium to vascular injury and may help explain why long-term responses in DES remain suboptimal.

The WAVE complex associates with sites of saddle membrane curvature.

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

Puerarin Attenuates LPS-Induced Inflammatory Responses and Oxidative Stress Injury in Human Umbilical Vein Endothelial Cells through Mitochondrial Quality Control.

Atherosclerosis is closely associated with the inflammatory reaction of vascular endothelial cells. Puerarin (Pue), the main active component isolated from the rhizome of Pueraria lobata, is an isoflavone compound with potent antioxidant properties. Although Pue exhibits promising antiatherosclerotic pharmacological effects, only a few studies have reported its protective effect on endothelial cells. This study found that Pue could partly regulate mitochondrial function in human umbilical vein endothelial cells (HUVECs) and reduce or inhibit lipopolysaccharide-induced inflammatory reactions and oxidative stress injury in HUVECs, likely via mitochondrial quality control. Furthermore, the protective effect of Pue on HUVECs was closely related to the SIRT-1 signaling pathway. Pue increased autophagy and mitochondrial antioxidant potential via increased SIRT-1 expression, reducing excessive production of ROS and inhibiting the expression of inflammatory factors and oxidative stress injury. Therefore, Pue may improve mitochondrial respiratory function and energy metabolism, increasing the vulnerability of HUVECs to an inflammatory state.

Electrospun polyurethane/poly (ɛ-caprolactone) nanofibers promoted the attachment and growth of human endothelial cells in static and dynamic culture conditions.

In this study, the angiogenic capacity of human endothelial cells was studied after being plated on the surface of polyurethane-poly caprolactone (PU/PCL) scaffolds for 72 h. In this study, cells were designated into five different groups, including PU, PU/PCL (2:1), PU/PCL (1:1); PU/PCL (1:2); and PCL. Data revealed that the PU/PCL (2:1) composition had a higher modulus and breakpoint in comparison with the other groups (p < 0.05). Compared to the other groups, the PU/PCL scaffold with a molar ratio of 2:1 had lower the contact angle θ and higher tensile stress (p < 0.05). The mean size of the PU nanofibers was reduced after the addition of PCL (p < 0.05). Based on our data, the culture of endothelial cells on the surface of PU/PCL (2:1) did not cause nitrosative stress and cytotoxic effects under static conditions compared to cells plated on a conventional plastic surface (p > 0.05). Based on data from the static condition, we fabricated a tubular PU/PCL (2:1) construct for six-day dynamic cell culture inside loop air-lift bioreactors. Scanning electron microscopy showed the attachment of endothelial cells to the luminal surface of the PU/PCL scaffold. Cells were flattened and aligned under the culture medium flow. Immunofluorescence imaging showed the attachment of cells to the luminal surface indicated by blue nuclei on the luminal surface. These data demonstrated that the application of PU/PCL substrate could stimulate endothelial cells activity under static and dynamic conditions.

Propofol-induced MiR-20b expression initiates endogenous cellular signal changes mitigating hypoxia/re-oxygenation-induced endothelial autophagy in vitro.

Certain miRNAs can attenuate hypoxia/re-oxygenation-induced autophagic cell death reported in our previous studies, but how these miRNAs regulate the autophagy-related cellular signaling pathway in preventing cell death is largely unknown. In the current study, the autophagy-related miRNAs of hsa-miR-20b were investigated in an in vitro model of hypoxia/re-oxygenation-induced endothelial autophagic cell death. Of these, miR-20b was found to be the most important miRNA which targeted on the key autophagy kinase ULK1 and inhibited hypoxia/re-oxygenation injury-induced autophagy by decreasing both autophagosomes and LC3I to II transition rate and P62 degradation. These processes were reversed by the transfection of an miR-20b inhibitor. Re-expression of ULK1 restores miR-20b-inhibited autophagy. Propofol, a commonly used anesthetic, promoted miR-20b and METTL3 expression and attenuated endothelial autophagic cell death. The inhibited endogenous expression of miR-20b or silenced METTL3 diminished the protective effect of propofol and accentuated autophagy. Additionally, METTL3 knockdown significantly inhibited miR-20b expression but up-regulated pri-miR-20b expression. Together, our data shows that propofol protects against endothelial autophagic cell death induced by hypoxia/re-oxygenation injury, associated with activation of METTL3/miR-20b/ULK1 cellular signaling.

The MARCH6-SQLE Axis Controls Endothelial Cholesterol Homeostasis and Angiogenic Sprouting.

The endothelial monolayer forms a barrier between the lumen of blood vessels and the underlying tissues. Stable VE-cadherin-based adherens junctions are essential for maintaining this barrier, whereas their remodeling is required for angiogenesis in health and disease. Here, we position the ERAD-associated ubiquitin ligase MARCH6 as a determinant of angiogenic sprouting and barrier integrity through its ability to promote the degradation of the rate-limiting cholesterol biosynthetic enzyme squalene epoxidase (SQLE). Accordingly, MARCHF6 ablation in endothelial cells increases SQLE protein and cholesterol load. This leads to altered membrane order, disorganized adherens junctions, decreased endothelial barrier function, and impaired SQLE-dependent sprouting angiogenesis. Akin to MARCHF6 silencing, the overexpression of SQLE impairs angiogenesis. However, angiogenesis is also attenuated when SQLE is silenced, indicating that fine-tuning cholesterol biosynthesis is a determinant of healthy endothelial function. In summary, we propose a mechanistic link between regulation of cholesterol homeostasis by the MARCH6-SQLE axis and endothelial integrity and angiogenesis.

Oleic acid and derivatives affect human endothelial cell mitochondrial function and vasoactive mediator production.

BACKGROUND: Inhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed. RESULTS: Dose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F2α, a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression. CONCLUSIONS: Together, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Fabrication of a self-assembled honeycomb nanofibrous scaffold to guide endothelial morphogenesis.

Controlling angiogenesis within tissue engineered constructs remains a critical challenge, especially with regard to the guidance of pre-vascular network formation. Here, we aimed to regulate angiogenesis on a self-assembled honeycomb nanofibrous scaffold. Scaffolds with honeycombs patterns have several desirable properties for tissue engineering, including large surface area, high structural stability and good permeability. Furthermore, the honeycomb pattern resembles early vascular network formation. The self-assembly electrospinning approach to honeycomb scaffolds is a technically simple, rapid, and direct way to realize selective deposition of nanofibers. To evaluate cell compatibility, spreading, proliferation and tube formation, human umbilical vein endothelial cells (HUVECs) were cultured on honeycomb scaffolds, as well as on random scaffolds for comparison. The optimized honeycomb nanofibrous scaffolds were observed to better support cell proliferation and network formation, which can facilitate angiogenesis. Moreover, HUVECs cultured on the honeycomb scaffolds were observed to reorganize their cell bodies into tube-like structures containing a central lumen, while this was not observed on random scaffolds. This work has shown that the angiogenic response can be guided by honeycomb scaffolds, allowing improved early HUVECs organization. The guided organization via honeycomb scaffolds can be utilized for tissue engineering applications that require the formation of microvascular networks.

High-Resolution Imaging of Intracellular Calcium Fluctuations Caused by Oscillating Microbubbles.

Ultrasound insonification of microbubbles can locally enhance drug delivery, but the microbubble-cell interaction remains poorly understood. Because intracellular calcium (Cai2+) is a key cellular regulator, unraveling the Cai2+ fluctuations caused by an oscillating microbubble provides crucial insight into the underlying bio-effects. Therefore, we developed an optical imaging system at nanometer and nanosecond resolution that can resolve Cai2+ fluctuations and microbubble oscillations. Using this system, we clearly distinguished three Cai2+ uptake profiles upon sonoporation of endothelial cells, which strongly correlated with the microbubble oscillation amplitude, severity of sonoporation and opening of cell-cell contacts. We found a narrow operating range for viable drug delivery without lethal cell damage. Moreover, adjacent cells were affected by a calcium wave propagating at 15 µm/s. With the unique optical system, we unraveled the microbubble oscillation behavior required for drug delivery and Cai2+ fluctuations, providing new insight into the microbubble-cell interaction to aid clinical translation.

Integrated super resolution fluorescence microscopy and transmission electron microscopy.

In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy. CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided. Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm.

Human lung microvascular endothelial cells as potential alternatives to human umbilical vein endothelial cells in bio-3D-printed trachea-like structures.

BACKGROUND: We have been trying to produce scaffold-free structures for airway regeneration using a bio-3D-printer with spheroids, to avoid scaffold-associated risks such as infection. Previous studies have shown that human umbilical vein endothelial cells (HUVECs) play an important role in such structures, but HUVECs cannot be isolated from adult humans. The aim of this study was to identify alternatives to HUVECs for use in scaffold-free structures. METHODS: Three types of structure were compared, made of chondrocytes and mesenchymal stem cells with HUVECs, human lung microvascular endothelial cells (HMVEC-Ls), and induced pluripotent stem cell (iPSC)-derived endothelial cells. RESULTS: No significant difference in tensile strength was observed between the three groups. Histologically, some small capillary-like tube formations comprising CD31-positive cells were observed in all groups. The number and diameters of such formations were significantly lower in the iPSC-derived endothelial cell group than in other groups. Glycosaminoglycan content was significantly lower in the iPSC-derived endothelial cell group than in the HUVEC group, while no significant difference was observed between the HUVEC and HMVEC-L groups. CONCLUSIONS: HMVEC-Ls can replace HUVECs as a cell source for scaffold-free trachea-like structures. However, some limitations were associated with iPSC-derived endothelial cells.

Glycosaminoglycan disaccharide compositional analysis of cell-derived extracellular matrices using liquid chromatography-tandem mass spectrometry.

Cell-derived extracellular matrices have emerged as promising scaffolds for tissue engineering (TE) strategies due to their ability to create a biomimetic microenvironment providing biochemical and physical cues to cells, without the limitations of availability and potential pathogen transmission associated with tissue-derived extracellular matrix (ECM) scaffolds. Glycosaminoglycans (GAGs) are important components of ECM with a crucial role in the maintenance of the mechanical properties of the tissue and as signaling regulators of several cellular processes, such as cell adhesion, growth and differentiation. However, despite their relevance to the field of TE, little information is available on the GAG composition of cell-derived ECM, mainly due to the lack of appropriate quantitative tools to determine different GAG and disaccharide subtypes in complex biological samples. In this chapter, we describe a highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to characterize decellularized cell-derived ECM generated in vitro in terms of their GAG and disaccharide composition.

Design of a Versatile Sample Holder for Facile Culture of Cells on Electrospun Membranes or Thin Polymer Films Under Flow Conditions.

Endothelial cell culture under flow, to mimic physiological conditions within blood vessels, has gained particular attention for the formation of a homogeneous endothelium in vitro. Here, we report on the design of a setup for simultaneous culture of up to nine electrospun membranes or thin polymer films in custom-made holders under flow on an orbital shaker. The versatile design of the device allows for the use of electrospun membranes/polymer films of choice and subsequent analysis with commonly used methods such as immunofluorescence or scanning electron microscopy.

The mechanisms of PM2.5 and its main components penetrate into HUVEC cells and effects on cell organelles.

Atmospheric particulate matter (PM2.5) is associated with the morbidity and mortality of cardiovascular diseases. However, whether PM2.5 penetrates into the cells and the potential mechanisms are unknown. Hence, the study firstly indicated that PM2.5 could penetrate into the HUVEC cells, and phagocytosis, micropinocytosis, caveolin as well as clathrin mediated the internalization of PM2.5 into HUVEC cells. Particularly, the components of PM2.5-Metal, PAHs and WSC could enter into HUVEC cells mainly via the micropinocytosis, clathrin and caveolin mediated endocytosis, respectively. The current data of environmental assessments indicated that PM2.5-Metal were extremely harmful to the ecological environment and human health. Moreover, accompanying with mitochondrial fusion gene Mfn1 was increased and fission genes Opa1 and Drp1 were decreased, and the lysosome related genes LAMP2 and LAMP3 were decreased, the phenomenon that the morphology of mitochondrial and lysosome injured was observed in HUVEC cells treated with PM2.5 and/or PM2.5-Metal. These data suggest that PM2.5 and its main components depend on different endocytosis penetrate into HUVEC cells and cause the mitochondrial and lysosomal damages. Thereby, our study provides the potential mechanism of haze particles penetration into HUVEC cells and damage to organelles.

The Interplay Between Cell-Cell and Cell-Matrix Forces Regulates Cell Migration Dynamics.

Cells in vivo encounter and exert forces as they interact with the extracellular matrix (ECM) and neighboring cells during migration. These mechanical forces play crucial roles in regulating cell migratory behaviors. Although a variety of studies have focused on describing single-cell or the collective cell migration behaviors, a fully mechanistic understanding of how the cell-cell (intercellular) and cell-ECM (extracellular) traction forces individually and cooperatively regulate single-cell migration and coordinate multicellular movement in a cellular monolayer is still lacking. Here, we developed an integrated experimental and analytical system to examine both the intercellular and extracellular traction forces acting on individual cells within an endothelial cell colony as well as their roles in guiding cell migratory behaviors (i.e., cell translation and rotation). Combined with force, multipole, and moment analysis, our results revealed that traction force dominates in regulating cell active translation, whereas intercellular force actively modulates cell rotation. Our findings advance the understanding of the intricacies of cell-cell and cell-ECM forces in regulating cellular migratory behaviors that occur during the monolayer development and may yield deeper insights into the single-cell dynamic behaviors during tissue development, embryogenesis, and wound healing.

Extracellular MicroRNA-92a Mediates Endothelial Cell-Macrophage Communication.

OBJECTIVE: Understanding message delivery among vascular cells is essential for deciphering the intercellular communications in cardiovascular diseases. MicroRNA (miR)-92a is enriched in endothelial cells (ECs) and circulation under atheroprone conditions. Macrophages are the primary immune cells in atherosclerotic lesions that modulate lesion development. Therefore, we hypothesize that, in response to atheroprone stimuli, ECs export miR-92a to macrophages to regulate their functions and enhance atherosclerotic progression. Approach and Results: We investigated the macrophage functions that are regulated by EC miR-92a under atheroprone microenvironments. We first determined the distributions of functional extracellular miR-92a by fractionating the intravesicular and extravesicular compartments from endothelial conditioned media and mice serum. The results indicate that extracellular vesicles are the primary vehicles for EC miR-92a transportation. Overexpression of miR-92a in ECs enhanced the proinflammatory responses and low-density lipoprotein uptake, while impaired the migration, of cocultured macrophage. Opposite effects were found in macrophages cocultured with ECs with miR-92a knockdown. Further analyses demonstrated that intravesicular miR-92a suppressed the expression of target gene KLF4 (Krüppel-like factor 4) in macrophages, suggesting a mechanism by which intravesicular miR-92a regulates recipient cell functions. Indeed, the overexpression of KLF4 rescued the EC miR-92a-induced macrophage atheroprone phenotypes. Furthermore, an inverse correlation of intravesicular miR-92a in blood serum and KLF4 expression in lesions was observed in atherosclerotic animals, indicating the potential function of extracellular miR-92a in regulating vascular diseases. CONCLUSIONS: EC miR-92a can be transported to macrophages through extracellular vesicles to regulate KLF4 levels, thus leading to the atheroprone phenotypes of macrophage and, hence, atherosclerotic lesion formation.

A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies.

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition.

A mathematical model of tumor-endothelial interactions in a 3D co-culture.

Intravasation and extravasation of cancer cells through blood/lymph vessel endothelium are essential steps during metastasis. Successful invasion requires coordinated tumor-endothelial crosstalk, utilizing mechanochemical signaling to direct cytoskeletal rearrangement for transmigration of cancer cells. However, mechanisms underlying physical interactions are difficult to observe due to the lack of experimental models easily combined with theoretical models that better elucidate these pathways. We have previously demonstrated that an engineered 3D in vitro endothelial-epithelial co-culture system can be used to isolate both molecular and physical tumor-endothelial interactions in a platform that is easily modeled, quantified, and probed for experimental investigation. Using this platform with mathematical modeling, we show that breast metastatic cells display unique behavior with the endothelium, exhibiting a 3.2-fold increase in interaction with the endothelium and a 61-fold increase in elongation compared to normal breast epithelial cells. Our mathematical model suggests energetic favorability for cellular deformation prior to breeching endothelial junctions, expending less energy as compared to undeformed cells, which is consistent with the observed phenotype. Finally, we show experimentally that pharmacological inhibition of the cytoskeleton can disrupt the elongatation and alignment of metastatic cells with endothelial tubes, reverting to a less invasive phenotype.