The Effect of High and Variable Glucose on the Viability of Endothelial Cells Co-Cultured with Smooth Muscle Cells.

Diabetes mellitus causes endothelial dysfunction. The aim of this study was to investigate the effect of normal (5 mmol/L), high (20 mmol/L), and fluctuating (5 and 20 mmol/L changed every day) glucose concentration in the culture medium on the viability of human umbilical vein endothelial cells (HUVECs) co-cultured with human umbilical artery smooth muscle cells (HUASMCs). The cultures were conducted on semi-permeable flat polysulfone (PSU) fibronectin-coated membranes immobilized in self-made inserts. The insert contained either HUVECs on a single membrane or HUASMCs and HUVECs on two membranes close to each other. Cultures were conducted for 7 or 14 days. Apoptosis, mitochondrial potential, and the production of reactive oxygen species and lactate by HUVECs were investigated. The results indicate that fluctuations in glucose concentration have a stronger negative effect on HUVECs viability than constant high glucose concentration. High and fluctuating glucose concentrations slow down cell proliferation compared to the culture carried out in the medium with normal glucose concentration. In conclusion, HUASMCs affect the viability of HUVECs when both types of cells are co-cultured in medium with normal or variable glucose concentration.

Transcription Factor Prospero Homeobox 1 (PROX1) as a Potential Angiogenic Regulator of Follicular Thyroid Cancer Dissemination.

It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes' expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.

Response of human normal and leukemia cells to factors released by amnion fragments in vitro.

Amnion is a membrane surrounding the embryo/fetus which determine growth factors and interleukins with angiogenic, immunogenic, and anti-inflammatory properties. The aim of the present study was to investigate the effects of conditioned culture medium from 24-h cultures of human amnion (hAM CCM) on migration and proliferation of human umbilical vein endothelial primary cells (HUVECs), freshly isolated bone marrow mononuclear cells (BM MNCs), and Jurkat leukemia cell line. Amnion membrane was freshly isolated from healthy placenta and its fragments cultured in vitro to produce hAM CCM. Members of the IGFBP protein family made up one third of all assayed proteins present in the hAM medium. The hAM CCM did not affect the proliferation rate of HUVECs or MNCs, but we observed more intensive migration of those cells, and lower expression of CD31 surface antigen on HUVECs as compared to control cultures. In contrast, Jurkat cells did not respond to hAM CCM treatment by proliferation or mobility change. The conditioned medium from 24-h cultures of human amnion is easy to obtain and is a convenient source of various growth and other factors that may be useful in practical medicine.

Human endothelial cells hollow fiber membrane bioreactor as a model of the blood vessel for in vitro studies.

Human endothelial cells are used in experimental models for studying in vitro pathophysiological mechanisms of different diseases. We developed an original bioreactor, which can simulate human blood vessel, with capillary polysulfone membranes covered with the human umbilical vein endothelial cells (HUVECs) and we characterized its properties. The elaborated cell seeding and culturing procedures ensured formation of a confluent cell monolayer on the inside surface of capillaries within 24 h of culturing under the shear stress of 6.6 dyn/cm(2). The optimal density of cells to be seeded was 60,000 cells/cm(2). Labeling HUVECs with carboxyfluorescein succinimidyl ester (CFSE) did not influence cells' metabolism. Flow cytometry-based analysis of HUVECs stained with CFSE demonstrated that in a presence of the shear stress cells' proliferation was much inhibited (after 72 h proliferation index was equal to 1.9 and 6.2 for cultures with and without shear stress, respectively) and the monolayer was formed mainly due to migration and spreading of cells that were physiologically elongated in a direction of the flow. Monitoring of cells' metabolism showed that HUVECs cultured in a presence of the shear stress preferred anaerobic metabolism and they consumed 1.5 times more glucose and produced 2.3 times more lactate than the cells cultured under static conditions. Daily von Willebrand factor production by HUVECs was near 2 times higher in a presence of the shear stress. The developed model can be used for at least 3 days in target studies under conditions mimicking the in vivo state more closely than the static HUVEC cultures.

Wound Area Measurement with Digital Planimetry: Improved Accuracy and Precision with Calibration Based on 2 Rulers.

INTRODUCTION: In the treatment of chronic wounds the wound surface area change over time is useful parameter in assessment of the applied therapy plan. The more precise the method of wound area measurement the earlier may be identified and changed inappropriate treatment plan. Digital planimetry may be used in wound area measurement and therapy assessment when it is properly used, but the common problem is the camera lens orientation during the taking of a picture. The camera lens axis should be perpendicular to the wound plane, and if it is not, the measured area differ from the true area. RESULTS: Current study shows that the use of 2 rulers placed in parallel below and above the wound for the calibration increases on average 3.8 times the precision of area measurement in comparison to the measurement with one ruler used for calibration. The proposed procedure of calibration increases also 4 times accuracy of area measurement. It was also showed that wound area range and camera type do not influence the precision of area measurement with digital planimetry based on two ruler calibration, however the measurements based on smartphone camera were significantly less accurate than these based on D-SLR or compact cameras. Area measurement on flat surface was more precise with the digital planimetry with 2 rulers than performed with the Visitrak device, the Silhouette Mobile device or the AreaMe software-based method. CONCLUSION: The calibration in digital planimetry with using 2 rulers remarkably increases precision and accuracy of measurement and therefore should be recommended instead of calibration based on single ruler.

Validation of a hemoglobin A1c model in patients with type 1 and type 2 diabetes and its use to go beyond the averaged relationship of hemoglobin A1c and mean glucose level.

BACKGROUND: Glycated hemoglobin A1c (HbA1c) has been used as an index of glycemic control in the management, guidance, and clinical trials of diabetic patients for the past 35 years. The aim of this study was to validate the HbA1c model in patients with type 1 and type 2 diabetes and to use it to support interpretation of HbA1c in different clinical situations. METHODS: The HbA1c model was identified in 30 patients (15 with type 1 diabetes and 15 with type 2 diabetes) by estimating the overall glycation rate constant (k), based on results of continuous glucose monitoring. The model was validated by assessing its ability to predict HbA1c changes in cultures of erythrocytes in vitro and to reproduce results of the A1C-Derived Average Glucose (ADAG) study. The model was used to simulate the influence of different glucose profiles on HbA1c. RESULTS: The mean k was equal to 1.296 ± 0.216 × 10(-9) l mmol(-1) s(-1) with no difference between type 1 and type 2 diabetes. The mean coefficient of variation of k was equal to 16.7%. The model predicted HbA1c levels in vitro with a mean absolute difference less than 0.3% (3.3 mmol/mol). It reproduced the linear relationship of HbA1c and mean glucose levels established in the ADAG study. The simulation experiments demonstrated that during periods of unstable glycemic control, glycemic profiles with the same mean glucose might result in much different HbA1c levels. CONCLUSIONS: Patients with type 1 and type 2 diabetes are characterized by the same mean value of k, but there is considerable interindividual variation in the relationship of HbA1c and mean glucose level. Results suggest that reciprocal changes in glycation rate and the life span of erythrocytes exist in a wide range of HbA1c values. Thus, for an average patient with diabetes, no modifications of parameters of the glycation model are required to obtain meaningful HbA1c predictions. Interpreting HbA1c as a measure of the mean glucose is fully justified only in the case of stable glycemia. The model and more frequent tests of HbA1c might be used to decrease ambiguity of interpreting HbA1c in terms of glycemic control.

Growth factors and their receptors derived from human amniotic cells in vitro.

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine.

Microdialysis monitoring of glucose, lactate, glycerol, and pyruvate in patients with diabetic ketoacidosis.

PURPOSE: The objective was to assess glucose, lactate, glycerol, and pyruvate concentrations in the interstitial fluid of the adipose tissue as well as the glucose relative recovery coefficient in reference to capillary blood (RC) during the first two days of the standard treatment of diabetic ketoacidosis (DKA) in patients with type 1 and type 2 diabetes. MATERIALS AND METHODS: The study group consisted of 19 patients (12 with type 1 diabetes and 7 with type 2 diabetes). The metabolic state of the patients was monitored using the microdialysis technique. The analysis of variance was used to investigate whether the type of diabetes and the duration of treatment influenced the assessed parameters. RESULTS: Concentrations of all the monitored components were stable after the initial 12 h of treatment. Glucose concentration was higher and concentrations of all the other components were lower (p<0.0001) in patients with type 1 diabetes than in patients with type 2 diabetes. Significantly higher RC was observed in patients with type 1 diabetes during the initial 12 h. CONCLUSIONS: The results suggest that the standard treatment of DKA is effective in stabilizing a concentration of the studied metabolic components in the interstitial fluid in patients with type 1 and type 2 diabetes despite differences in the glucose concentration at the beginning of the treatment.

Accuracy and precision of selected wound area measurement methods in diabetic foot ulceration.

AIM: The present study assesses precision and accuracy of four selected methods of wound area measurements in diabetic foot wounds. MATERIALS AND METHODS: The areas of 16 wound shapes were measured using linear measurement with elliptical estimation (Elliptical method), using the Visitrak™ (Smith & Nephew, London, United Kingdom) device, the SilhouetteMobile™ device (ARANZ Medical Ltd., Christchurch, New Zealand), and the TeleDiaFoS system (Nalecz Institute of Biocybernetics and Biomedical Engineering, Warsaw, Poland). The actual area of a wound shape was determined with a tested scanner and specifically developed software. Accuracy of the area measurement method was assessed by the relative error (RE), whereas precision was assessed by the coefficient of variation (CV). RESULTS: The overall absolute REs were 13.3%, 6.8%, 2.1%, and 2.3% for the Elliptical method, the Visitrak device, the TeleDiaFoS system, and the SilhouetteMobile device, respectively. The accuracy of the Visitrak device was remarkably reduced for wound areas smaller than 2 cm². The overall CVs were 6.0%, 6.3%, 1.6%, and 3.1% for the Elliptical method, the Visitrak device, the TeleDiaFoS system, and the SilhouetteMobile device, respectively. The precision of the Visitrak device was revealed to be remarkably lower for small wounds (<2 cm²). CONCLUSIONS: The Elliptical method gives overestimation up to 33%; thus, it should not be used in applications where the actual wound area is an important parameter (like the prediction of wound healing). The TeleDiaFoS system and the SilhouetteMobile device showed the best accuracy of all used methods; however, the precision of the TeleDiaFoS system was revealed to be higher than the precision of the SilhouetteMobile device. The accuracy and the precision of the Visitrak device are significantly reduced for wounds smaller than 2 cm².

Hemoglobin glycation rate constant in non-diabetic Individuals.

The objectives were as follows: (1) estimating mean value of the overall hemoglobin glycation rate constant (k); (2) analyzing inter-individual variability of k; (3) verifying ability of the hemoglobin A1c (HbA1c) formation model to predict changes of HbA1c during red blood cells cultivation in vitro and to reproduce the clinical data. The mean k estimated in a group of 10 non-diabetic subjects was equal to 1.257 ± 0.114 × 10(-9) L mmol(-1) s(-1). The mean k was not affected by a way of estimation of glycemia. The mean k differed less than 20% from values reported earlier and it was almost identical to the mean values calculated on basis of the selected published data. Analysis of variability of k suggests that inter-individual heterogeneity of HbA1c formation is limited or rare. The HbA1c mathematical model was able to predict changes of HbA1c in vitro resulting from different glucose levels and to reproduce a linear relationship of HbA1c and average glucose obtained in the A1C-Derived Average Glucose Study. This study demonstrates that the glycation model with the same k value might be used in majority of individuals as a tool supporting interpretation of HbA1c in different clinical situations.

A new imaging and data transmitting device for telemonitoring of diabetic foot syndrome patients.

BACKGROUND: Proper healing of ulcers and wounds on the feet of diabetes patients is important in order to prevent amputation. If the wound area reduction during the first 4 weeks of the treatment is not 40% or more, reevaluation of the treatment is necessary. The wound area evaluation is not complicated when the patient stays at a hospital, but when he or she goes home the physician does not have a tool allowing monitoring of the wound area. METHODS AND RESULTS: The aim of the present article is to present a new device able to take a wound picture and send it automatically to the database. This device, called the Patient's Module (PM), is also able to download data from the memories of blood pressure and blood glucose meters and send the data to the database. The PM is able to operate within the TeleDiaFoS system (developed earlier in collaboration with the Department and Clinic of Gastroenterology and Metabolic Diseases, Medical University of Warsaw, Warsaw, Poland) aimed at monitoring of treatment of patients with diabetic foot syndrome. The PM was tested on 10 type 2 diabetes patients during a 3-month period. CONCLUSIONS: The study revealed that the PM can be used as a home telemonitoring device, and the analysis of the data sent from patient's home enables the assessment of wound healing progress, giving the physician the possibility for earlier change of the treatment if the wound area reduction is not satisfactory.

Validation of hemoglobin glycation models using glycemia monitoring in vivo and culturing of erythrocytes in vitro.

Glycated hemoglobin A1c (HbA1c) concentration in blood is an index of the glycemic control widely used in diabetology. The aim of the work was to validate two mathematical models of HbA1c formation (assuming irreversible or reversible glycation, respectively) and select a model, which was able to predict changes of HbA1c concentration in response to varying glycemia courses with higher accuracy. The experimental procedure applied consisted of an original combination of: in vivo continuous glucose concentration monitoring, long-term in vitro culturing of the human erythrocytes and mathematical modeling of HbA1c formation in vivo and in vitro with HbA1c values scaled according to the most specific analytical methods. Sixteen experiments were conducted in vitro using blood samples collected from healthy volunteer and stable type 1 diabetic patients whose glycemia was estimated beforehand based on long-term monitoring. The mean absolute difference of the measured and predicted HbA1c concentrations for the in vitro experiments were equal to 0.64 +/- 0.29% and 1.42 +/- 0.16% (p = 0.0007) for irreversible and for reversible model, respectively, meaning that the irreversible model was able to predict the glycation kinetics with a higher accuracy. This model was also more sensitive to a deviation of the erythrocytes life span.

Microdialysis technique as a monitoring system for acute complications of diabetes.

The objective of the study was to establish the quasi-continuous courses, using microdialysis technique, of glucose, lactate, and glycerol concentrations in interstitial fluid of abdominal adipose tissue during the standard treatment of acute diabetes complications. Clinical studies were carried out on 31 diabetic patients during the initial 48 h of the treatment. In all but two obese female patients with hyperglycemic hyperosmolar state (HHS) did glucose concentration in perfusion fluid (PF) reflect concentration in capillary blood. The recovery of glucose correlated with patients' body mass index (r = 0.55). It was significantly higher in lean and overweight patients (91 +/- 15%) than in obese patients (55 +/- 31%). The course of lactate concentration in PF coincided with the course in venous blood (2.1 +/- 0.3 mmol/L vs. 2.0 +/- 0.5 mmol/L, P = 0.35). Glycerol concentration was 267 +/- 41 micromol/L and 133 +/- 40 micromol/L in PF and venous blood, respectively (P = 0.004). The study indicated that microdialysis may be an effective tool to monitor concentration of different metabolites in interstitial fluid of the adipose tissue during treatment of the acute complications of diabetes. Applicability of the technique in the monitoring of HHS, especially in obese female patients, needs further investigation.

Impact of convective transport on dialyzer clearance.

The results of predictions of three mathematical models used to describe the impact of convective flow on dialyzer clearance are presented. These models are based on the ordinary differential equations, which describe changes of solute concentration and solute and fluid flows along the module length. One of the models takes into consideration the existence of the boundary layers on both sides of the membrane wall, by including in the equations two parameters kB and kD, which describe mass transport coefficients in blood and dialysate, respectively. In the second model, the boundary layers are included in one lumped membrane permeability parameter. The diffusive membrane permeability was calculated from pure diffusive clearance, which was taken from experimental results. In the third model, a linear dependence of transmittance coefficient and ultrafiltration flow was proposed. The theoretical results were compared with data obtained in experiments carried out in vitro with four types of high-flux hollow-fiber dialyzers. The comparisons demonstrate that the first two models are of similar accuracy and the third model is not suitable for small solutes.

Polypropylene hollow fiber for cells isolation: methods for evaluation of diffusive transport and quality of cells encapsulation.

Formulation of membrane properties is important prior the successful implantation of encapsulated cells producing therapeutically relevant compounds. The purpose of our study was to specify the methods allowing preliminary evaluation of hollow fibers (HF) chosen for immunoisolation. We have selected as estimates (1) diffusive permeability for small and large solutes, and HF cut off (in vitro), (2) histological evaluation of tissue overgrowth after sc. implantation into mice. It was found that diffusive coefficients were linearly dependent on the particle diameter except that of albumin (2-3 times higher than theoretically estimated). This discrepancy imply that for certain particles the interaction with membrane material may be significant. The histological evaluation showed that siliconized HF implanted for 105 days were accepted (there was thin fibrotic layer on the external surface of the HF, no surrounding haemopoietic cells were found). It is concluded that proposed methods for preliminary evaluation of hollow fibers chosen for immunoisolation seems to be reliable and suitable for testing diffusive permeability of each relevant cell product.