The intriguing role of collagen on the rheology of cancer cell spheroids.

Spheroids are multicellular systems with an interesting rheology giving rise to elasto-visco-plastic properties. They are good tumor models, but the role of the extracellular matrix (ECM) is not fully understood. ECM is an important link between cells and may have a significant impact on tissue organization. Here we determine viscoelastic properties of spheroids including different collagen I amounts using AFM and predict new frequency-dependent properties leading to soft glassy rheology behavior. A unified model - similar to single cell behavior - is proposed and discussed, while complementary confocal experiments reveal the microstructure of spheroids, with collagen I fibers serving as a skeleton for cells, thus reinforcing the spheroid viscoelastic behavior.

The effect of shear stress reduction on endothelial cells: A microfluidic study of the actin cytoskeleton.

Reduced blood flow, as occurring in ischemia or resulting from exposure to microgravity such as encountered in space flights, induces a decrease in the level of shear stress sensed by endothelial cells forming the inner part of blood vessels. In the present study, we use a microvasculature-on-a-chip device in order to investigate in vitro the effect of such a reduction in shear stress on shear-adapted endothelial cells. We find that, within 1 h of exposition to reduced wall shear stress, human umbilical vein endothelial cells undergo reorganization of their actin skeleton with a decrease in the number of stress fibers and actin being recruited into the cells' peripheral band, indicating a fairly fast change in the cells' phenotype due to altered flow.

A biomechanical model for the transendothelial migration of cancer cells.

We propose a biomechanical model for the extravasation of a tumor cell (TC) through the endothelium of a blood vessel. Based on prior in vitro observations, we assume that the TC extends a protrusion between adjacent endothelial cells (ECs) that adheres to the basement membrane via focal adhesions (FAs). As the protrusion grows in size and branches out, the actomyosin contraction along the stress fibers (SFs) inside the protrusion pulls the relatively rigid nucleus through the endothelial opening. We model the chemo-mechanics of the SFs and the FAs by following the kinetics of the active myosin motors and high-affinity integrins, subject to mechanical feedback. This is incorporated into a finite-element simulation of the extravasation process, with the contractile force pulling the nucleus of the TC against elastic resistance of the ECs. To account for the interaction between the TC nucleus and the endothelium, we consider two scenarios: solid-solid contact and lubrication by cytosol. The former gives a lower bound for the required contractile force to realize transmigration, while the latter provides a more realistic representation of the process. Using physiologically reasonable parameters, our model shows that the SF and FA ensemble can produce a contractile force on the order of 70 nN, which is sufficient to deform the ECs and enable transmigration. Furthermore, we use an atomic force microscope to measure the resistant force on a human bladder cancer cell that is pushed through an endothelium cultured in vitro. The magnitude of the required force turns out to be in the range of 70-100 nN, comparable to the model predictions.

Paroxysmal Permeability Disorders: Development of a Microfluidic Device to Assess Endothelial Barrier Function.

Background: Paroxysmal Permeability Disorders (PPDs) are pathological conditions caused by periodic short lasting increase of endothelial permeability, in the absence of inflammatory, degenerative, ischemic vascular injury. PPDs include primary angioedema, idiopathic systemic capillary leak syndrome and some rare forms of localized retroperitoneal-mediastinal edema. Aim: to validate a microfluidic device to study endothelial permeability in flow conditions. Materials and Methods: we designed a microchannel network (the smallest channel is 30µm square section). Human Umbilical Vein Endothelial Cells (HUVECs) were cultured under constant shear stress in the networks. Endothelial permeability assessment was based on interaction of biotinylated fibronectin used as a matrix for HUVECs and FITC-conjugated avidin. The increase in endothelial permeability was identified as changes in fluorescence intensity detected by confocal fluorescent microscopy. Results: The microchannels were constantly perfused with a steady flow of culture medium, ensuring a physiologically relevant level of shear stress at the wall of ~0.2 Pa. Our preliminary results demonstrated that circulation of culture medium or plasma from healthy volunteers was associated with low fluorescence of fibronectin matrix. When bradykinin diluted in culture medium was perfused, an increase in average fluorescence was detected. Conclusion: Our microvasculature model is suitable to study endothelial functions in physiological flow conditions and in the presence of factors like bradykinin known as mediator of several PPDs. Therefore, it can be a promising tool to better understand the mechanisms underlying disorders of endothelial permeability.

Gestation age-associated dynamics of mitochondrial calcium uniporter subunits expression in feto-maternal complex at term and preterm delivery.

Calcium plays a role of universal cellular regulator in the living cell and one of the crucial regulators of proper fetal development during gestation. Mitochondria are important for intracellular calcium handling and signaling. Mitochondrial calcium uniporter (mtCU) is a multiprotein complex of the mitochondrial inner membrane responsible for the transport of calcium to the mitochondrial matrix. In the present study, we analyzed the expression level of mtCU components in two parts of the feto-maternal system - placenta and myometrium at full-term delivery and at preterm birth (PTB) on different stages: 22-27, 28-32, 33-36 weeks of gestation (n = 50). A gradual increase of mRNA expression and changes in protein content of MCU and MICU1 subunits were revealed in the placenta during gestation. We also observed slower depolarization rate of isolated placental mitochondria induced by Ca2+ titration at PTB. In myometrium at PTB relative gene expression level of MCU, MCUb and SMDT1 increased as compared to full-term pregnancy, but the tendency to gradual increase of MCU protein simultaneous with MCUb increase and MICU1 decline was shown in gestational dynamics. Changes observed in the present study might be considered both natural dynamics as well as possible pathological mechanisms underlying preterm birth.

Cumulus cell mitochondrial activity in relation to body mass index in women undergoing assisted reproductive therapy.

Most studies have considered the negative influence of obesity on fertility in both genders. In the present study, we assessed mitochondrial activity expressed as the mitochondrial potential index (MPI) in cumulus cells from obese women and women with a normal body mass index (BMI) during assisted reproductive therapy. The results revealed a significant reduction of MPI with increased body mass. The lower MPI levels in cumulus cells from obese women may reflect mitochondrial dysfunction caused by oxidative stress, which can affect the cumulus-oocyte complex and have an impact on oocyte development.

BION-M 1: First continuous blood pressure monitoring in mice during a 30-day spaceflight.

Animals are an essential component of space exploration and have been used to demonstrate that weightlessness does not disrupt essential physiological functions. They can also contribute to space research as models of weightlessness-induced changes in humans. Animal research was an integral component of the 30-day automated Russian biosatellite Bion-M 1 space mission. The aim of the hemodynamic experiment was to estimate cardiovascular function in mice, a species roughly 3000 times smaller than humans, during prolonged spaceflight and post-flight recovery, particularly, to investigate if mice display signs of cardiovascular deconditioning. For the first time, heart rate (HR) and blood pressure (BP) were continuously monitored using implantable telemetry during spaceflight and recovery. Decreased HR and unchanged BP were observed during launch, whereas both HR and BP dropped dramatically during descent. During spaceflight, BP did not change from pre-flight values. However, HR increased, particularly during periods of activity. HR remained elevated after spaceflight and was accompanied by increased levels of exercise-induced tachycardia. Loss of three of the five mice during the flight as a result of the hardware malfunction (unrelated to the telemetry system) and thus the limited sample number constitute the major limitation of the study. For the first time BP and HR were continuously monitored in mice during the 30-day spaceflight and 7-days of post-flight recovery. Cardiovascular deconditioning in these tiny quadruped mammals was reminiscent of that in humans. Therefore, the loss of hydrostatic pressure in space, which is thought to be the initiating event for human cardiovascular adaptation in microgravity, might be of less importance than other physiological mechanisms. Further experiments with larger number of mice are needed to confirm these findings.

Microvasculature on a chip: study of the Endothelial Surface Layer and the flow structure of Red Blood Cells.

Microvasculatures-on-a-chip, i.e. in vitro models that mimic important features of microvessel networks, have gained increasing interest in recent years. Such devices have allowed investigating pathophysiological situations involving abnormal biophysical interactions between blood cells and vessel walls. Still, a central question remains regarding the presence, in such biomimetic systems, of the endothelial glycocalyx. The latter is a glycosaminoglycans-rich surface layer exposed to blood flow, which plays a crucial role in regulating the interactions between circulating cells and the endothelium. Here, we use confocal microscopy to characterize the layer expressed by endothelial cells cultured in microfluidic channels. We show that, under our culture conditions, endothelial cells form a confluent layer on all the walls of the circuit and display a glycocalyx that fully lines the lumen of the microchannels. Moreover, the thickness of this surface layer is found to be on the order of 600 nm, which compares well with measurements performed ex or in vivo on microcapillaries. Furthermore, we investigate how the presence of endothelial cells in the microchannels affects their hydrodynamic resistance and the near-wall motion of red blood cells. Our study thus provides an important insight into the physiological relevance of in vitro microvasculatures.

Mitochondrial role in adaptive response to stress conditions in preeclampsia.

Preeclampsia (PE) is a pregnancy-specific syndrome, characterized in general by hypertension with proteinuria or other systemic disturbances. PE is the major cause of maternal and fetal morbidity and mortality worldwide. However, the etiology of PE still remains unclear. Our study involved 38 patients: 14 with uncomplicated pregnancy; 13 with early-onset PE (eoPE); and 11 with late-onset PE (loPE). We characterized the immunophenotype of cells isolated from the placenta and all biopsy samples were stained positive for Cytokeratin 7, SOX2, Nestin, Vimentin, and CD44. We obtained a significant increase in OPA1 mRNA and protein expression in the eoPE placentas. Moreover, TFAM expression was down-regulated in comparison to the control (p < 0.01). Mitochondrial DNA copy number in eoPE placentas was significantly higher than in samples from normal pregnancies. We observed an increase of maximum coupled state 3 respiration rate in mitochondria isolated from the placenta in the presence of complex I substrates in the eoPE group and an increase of P/O ratio, citrate synthase activity and decrease of Ca(2+)-induced depolarization rate in both PE groups. Our results suggest an essential role of mitochondrial activity changes in an adaptive response to the development of PE.