Cultures Of Endothelial Cells Research Articles

Chrysin boosts KLF2 expression through suppression of endothelial cell-derived exosomal microRNA-92a in the model of atheroprotection.

Atherosclerosis and its related clinical complications are the leading cause of death. MicroRNA (miR)-92a in the inflammatory endothelial dysfunction leads to atherosclerosis. Krüppel-like factor 2 (KLF2) is required for vascular integrity and endothelial function maintenance. Flavonoids possess many biological properties. This study investigated the vascular protective effects of chrysin in balloon-injured carotid arteries. Exosomes were extracted from human coronary artery endothelial cell (HCAEC) culture media. Herb flavonoids and chrysin were the treatments in these atheroprotective models. Western blotting and real-time PCRs were performed. In situ hybridization, immunohistochemistry, and immunofluorescence analyses were employed. MiR-92a increased after balloon injury and was present in HCAEC culture media. Chrysin was treated, and significantly attenuated the miR-92a levels after balloon injury, and similar results were obtained in HCAEC cultures in vitro. Balloon injury-induced miR-92a expression, and attenuated KLF2 expression. Chrysin increased the KLF2 but reduced exosomal miR-92a secretion. The addition of chrysin and antagomir-92a, neointimal formation was reduced by 44.8 and 49.0% compared with balloon injury after 14days, respectively. Chrysin upregulated KLF2 expression in atheroprotection and attenuated endothelial cell-derived miR-92a-containing exosomes. The suppressive effect of miR-92a suggests that chrysin plays an atheroprotective role. Proposed pathway for human coronary artery endothelial cell (HCAEC)-derived exosomes induced by chrysin to suppress microRNA (miR)-92a expression and counteract the inhibitory effect of miR-92a on KLF2 expression in HCAECs. This provides an outline of the critical role of the herbal flavonoid chrysin, which may serve as a valuable therapeutic supplement for atheroprotection.

Heme cytotoxicity is the consequence of endoplasmic reticulum stress in atherosclerotic plaque progression

Hemorrhage and hemolysis with subsequent heme release are implicated in many pathologies. Endothelial cells (ECs) encounter large amount of free heme after hemolysis and are at risk of damage from exogenous heme. Here we show that hemorrhage aggravates endoplasmic reticulum (ER) stress in human carotid artery plaques compared to healthy controls or atheromas without hemorrhage as demonstrated by RNA sequencing and immunohistochemistry. In EC cultures, heme also induces ER stress. In contrast, if cultured ECs are pulsed with heme arginate, cells become resistant to heme-induced ER (HIER) stress that is associated with heme oxygenase-1 (HO-1) and ferritin induction. Knocking down HO-1, HO-2, biliverdin reductase, and ferritin show that HO-1 is the ultimate cytoprotectant in acute HIER stress. Carbon monoxide-releasing molecules (CORMs) but not bilirubin protects cultured ECs from HIER stress via HO-1 induction, at least in part. Knocking down HO-1 aggravates heme-induced cell death that cannot be counterbalanced with any known cell death inhibitors. We conclude that endothelium and perhaps other cell types can be protected from HIER stress by induction of HO-1, and heme-induced cell death occurs via HIER stress that is potentially involved in the pathogenesis of diverse pathologies with hemolysis and hemorrhage including atherosclerosis.

Possible role of WNT10B in increased proliferation and tubule formation of human umbilical vein endothelial cell cultures treated with hypoxic conditioned medium from human adipocytes

ABSTRACT Regulation of angiogenesis plays an important role in adipose tissue expansion and function. The Wnt pathway and WNT10B, the main member of Wnt family, participate in angiogenesis in cancer tumors, but there is limited evidence to support the regulatory role of WNT10B in human adipose tissue angiogenesis. Subcutaneous white adipose tissue (scWAT) of 80 participants including obese and non-obese subjects was obtained and the expression of WNT10B and VEGFA genes were evaluated using qPCR. Human adipose-derived stem cells (hADSC) were differentiated to adipocytes and incubated under either hypoxic or normoxic conditions. The conditioned media of these adipocytes were collected and used as growth media for human umbilical vein endothelial cells (HUVEC) in Matrigel. We evaluated the proliferation, cell cycle phases, tubule formation and β-catenin activation of these treated cells. We found a significant correlation between WNT10B and VEGFA expression in the scWAT of both obese and non-obese subjects. Proliferation and tubule formation of HUVEC treated with conditioned media of hypoxic adipocytes (hCM) in the S-phase were increased significantly compared to the HUVEC treated with the conditioned media of normoxic adipocytes (nCM). The expression of WNT10B and VEGFA was enhanced in hypoxic adipocytes compared to normoxic adipocytes; also, activation and nuclear translocation of β-catenin was enhanced in the HUVEC treated with hCM compared to nCM. WNT10B acts as an angiogenic protein in scWAT under hypoxic conditions. Hypoxia induced WNT10B increases VEGFA expression and causes tube formation by HUVECs and angiogenesis in adipose tissue via the canonical Wnt/β-catenin pathway.

Static and dynamic culture of human endothelial cells encapsulated inside alginate-gelatin microspheres

This study aimed to explore the angiogenesis potential of human endothelial cells encapsulated inside alginate-gelatin microspheres under static and dynamic culture systems after 7 days.Human umbilical vein endothelial cells were encapsulated inside alginate (1%) and gelatin (1.2%) using an electrostatic encapsulation method. Cells were incubated for 7 days in vitro. The cell survival rate was measured using the MTT assay. The expression of VEGFR-2 and von Willebrand factor genes was studied by real-time PCR assay. Using western blot analysis, we monitored the protein contents of VEGFR-2, vWF, and Caspase 3. The levels of SOD and GPx enzymes were calculated using biochemical kits. Angiogenesis potential was assessed using in vitro Matrigel assay.Data showed an increased survival rate in encapsulated cells cultured under the static condition compared to the conventional 2D condition (p < 0.05). The culture of encapsulated cells under a dynamic bioreactor system did not alter cell viability. Compared to the dynamic culture system, the incubation of encapsulated cells in the static culture system swelled the microspheres (p < 0.05). Both dynamic and static culture models increased the expression of VEGFR-2 and von Willebrand factor in encapsulated cells compared to 2D culture (p < 0.05), showing enhanced functional maturation. Data showed a significant increase of vWF and reduction of apoptosis marker Caspase in the dynamic culture system (p < 0.05). The levels of SOD and GPx were significantly increased in dynamic and static culture models as compared to the control 2D group (p < 0.05). In vitro tubulogenesis assay showed significant induction of angiogenesis in dynamic encapsulated HUVECs indicated with a large number of vascular tubes and arborized ECs compared to the control and static encapsulated HUVECs (p < 0.05).The current study suggests a bioreactor dynamic system is a reliable approach, similar to a static condition, for the expansion of encapsulated human ECs in a 3D milieu.

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

SARS‐CoV‐2 spike protein induces degradation of junctional proteins that maintain endothelial barrier integrity

BackgroundSARS‐CoV‐2 enters cells through the Angiotensin‐converting enzyme 2 (ACE‐2) receptor present on the cell surface. ACE‐2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. Reports suggest that COVID‐19 patients also showed symptoms related to endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. We investigated this in vitro using endothelial cell culture and recombinant SARS‐CoV‐2 spike protein S1 Receptor‐Binding Domain (S1RBD).Materials and MethodsMouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. The expression of VE‐Cadherin, PECAM‐1, JAM‐A and Connexin 43, was probed by Western blot in normal and diabetic cells, before and after treatment with S1RBD.ResultsThe expression of VE‐Cadherin, an endothelial adherens junction protein, was not affected by diabetes or by S1RBD treatment. PECAM‐1 expression showed a small but significant (~15%) decrease after S1RD treatment in control cells. Junctional Adhesion Molecule (JAM‐A), a tight junction protein, and the gap junction protein, Connexin‐43, decreased by ~50 and 80% respectively in the presence of S1RBD when compared to untreated controls. Expression of PECAM‐1, JAM‐A and connexin‐43 were significantly lower in untreated diabetic endothelial cells, compared to untreated control cells. This expression level further decreased significantly in the presence of S1RBD, with maximal effects observed with JAM‐A and Connexin‐43 expression (~80 and 90% decrease, respectively). These results indicate that healthy and diabetic endothelial cells respond differently when challenged with SARS‐CoV‐2 S1RBD.ConclusionS1RBD‐induces degradation of endothelial junctional proteins that likely affects endothelial barrier function and causes vascular damage.

On-chip electrocatalytic NO sensing using ruthenium oxide nanorods

Online, on-chip measurement of nitric oxide (NO) in organ-on-chip devices is desired to study endothelial (dys)function under dynamic conditions. In this work, ruthenium oxide (RuOx) is explored as an amperometric NO sensor and its suitability for organ-on-chip applications. For testing purposes, diethylamine NONOate was used as chemical NO donor. The NONOate’s NO generation and electrochemical oxidation of generated NO were confirmed by real-time electrochemical/mass-spectrometry. Using RuOx nanorods electrodes, we show that NO oxidation occurred at a lower onset potential (+675 mV vs. Ag/AgCl) than on bare Pt electrode (+800 mV vs. Ag/AgCl). Due to NO adsorption on the RuOx surface, NO oxidation also delivered a higher current density (33.5 nA.μM−1. cm-2) compared to bare Pt (19.6 nA.μM−1. cm-2), making RuOx nanorods a favourable electrode for NO sensing applications. The RuOx electrode’s suitability for organ-on-chip applications was successfully tested by using the electrode to detect a few micromolar concentration of NO generated by endothelial cell culture. Overall, the RuOx nanorods proved to be suitable for organ-on-chip studies due to their high sensitivity and selectivity. Our chip-integrated electrode allows for online NO monitoring in biologically relevant in vitro experiments.

Endothelial‐Cell Modifications in Murine Collecting Lymphatic Vessels in Obesity

The presence of edema within tissues and cavities presented by patients afflicted with obesity suggests that lymphatic dysfunction may be a contributing factor during the onset and/or development of the various ailments associated with this disease. Importantly, obesity has been shown to significantly increase the chances of developing type-2 diabetes, cardiovascular diseases, and lymphedema in breast cancer patients undergoing surgical intervention and radiotherapy. In obesity, vascular endothelial dysfunction is known to result as a consequence of the chronic systemic inflammation. Relevant to this study, the extent of lymphatic endothelial dysfunction in obesity and type-2 diabetes remains to be fully assessed. In order to 1) elucidate the root cause of lymphatic dysfunction and subsequent impaired lymph transport in obesity, and 2) determine if this dysfunction involves an endothelial component; it is essential to isolate the various cell types in the lymphatic wall and compare the specific genetic and proteomic makeup in health and disease. Here we focus on the study and understanding of the disease-driven modifications to lymphatic endothelial cells (LECs). We hypothesize that LECs of collecting lymphatic vessels from obese mice will exhibit differential expression of transcription factors commonly associated with endothelial dysfunction (i.e. LEC specification, valve dysfunction, and barrier disruption). We utilized a diet-induced obesity mouse model. Obesity was induced by feeding C57BL/6J male mice a western diet (ResearchDiets Inc. Cat.No. D12079Bi) for 14 weeks starting at 5 weeks of age. Consistent with previous studies, diet-induced obese animals gained twice as much weight when compared to aged-matched animals fed a control diet (weight gain 18.8 g vs 9.7 g). Non-fasted blood glucose levels were 41.6±10.6% higher in obese animals, which also displayed increased skin moisture content, 45.1±4.7% (obese) vs 32.6±4.8% (control), as determined by a wet-to-dry weight ratio. Our preliminary qRT-PCR results on fluorescence-activated cell sorting (FACS) purified LECs show that diet-induced obesity results in decreased expression of the transcription factor, and LEC marker, Prox1. We have developed a novel protocol for establishing cell cultures of primary LECs from collecting lymphatic vessels isolated from different species. Notably, the same protocol with minor modifications can be used to establish cultures of primary endothelial cells from blood vessels. Preliminary characterization of murine primary LECs using RT-PCR, immunofluorescence, and confocal microscopy suggests that these cultures could be a useful novel tool for lymphatic research. Currently, we are using these cultured primary LECs from db/db mice and a proteomics approach to further assess the effects of obesity and type-2 diabetes in the lymphatic endothelium of collecting lymphatics. We anticipate that these studies will provide critical information to identify novel mechanisms of lymphatic endothelial dysfunction in obesity, type-2 diabetes, and lymphedema, and propose novel therapeutic targets to ameliorate lymphatic dysfunction in these diseases.

Acetaldehyde in e‐cigarette aerosol and cigarette smoke exacerbate pulmonary microvascular endothelial dysfunction in ALDH2*2 mice

Background Pulmonary endothelial dysfunction caused by the frequent exposure to cigarette smoke can lead to pulmonary artery hypertension (PAH). E-cigarette vapor, similar to cigarette smoke, contains the aldehydes including acetaldehyde. Further, ~560 million people carry an inactivating genetic variant in aldehyde dehydrogenase 2 (ALDH2), known as ALDH2*2, the primary enzyme responsible for metabolizing acetaldehyde. However, little is known how reactive acetaldehyde present in cigarette smoke and e-cigarette aerosol, when coupled with genetics, influence pulmonary vascular function. Hypothesis: Acetaldehyde in e-cigarette aerosol and cigarette smoke lead to pulmonary endothelial dysfunction that is exacerbated in mice carrying the ALDH2*2 variant. Methods: Primary cultures of pulmonary microvascular endothelial cells from wild type ALDH2 and ALDH2*2 male mice were used. Cultures were treated with acetaldehyde (1 μM for 1 hour) in the presence and absence of the ALDH2 activator, Alda-1 (20 μM). Intracellular ROS accumulation, reactive aldehyde-induced protein adducts, mitochondrial bioenergetics, and endothelial function by capillary tube formation were measured. ANOVA with Bonferroni correction was performed for multiple comparisons between groups. * P<0.05. Results: In ALDH2 and ALDH2*2 mice, no differences were noted in endothelial cell morphology and expression of CD31, an endothelial specific marker. However, when exposed to acetaldehyde, pulmonary microvascular endothelial cells from ALDH2*2 mice showed a 91.2±23.2% increase in 4-HNE-induced protein adducts, a 27.8±5.1% reduction in mitochondrial bioenergetics, a 11.1±1.6% decrease in cell viability, and an 70.4±17.2% exacerbation of endothelial dysfunction compared to wild type ALDH2 cells (p<0.05 for each assay, n=3 biological replicates). Pretreatment with Alda-1 partially reversed the oxidative stress and endothelial injury caused by acetaldehyde treatment. Conclusion: Acetaldehyde, the primary aldehyde present in e-cigarette aerosol which is also in cigarette smoke triggers pulmonary endothelial dysfunction particularly for rodents carrying an inactivating ALDH2 variant, ALDH2*2. This study provides a mechanism how e-cigarette vapor similar to cigarette smoke can trigger endothelial dysfunction that can over time potentially lead to the development of PAH.

Tunicamycin interferes with WNT signaling and inhibits angiogenesis

Wnt signaling pathway is a family of proteins that can lead to a cascade of cell proliferation. The transcription co-activator β-catenin has been associated with the development of tissue. Recent studies have shown that Wnt signaling have a significant role in vascular development, an important event in tumor growth. Therefore, it regulates some biological processes (i.e. cell proliferation, apoptosis). Our laboratory has established that protein N-glycosylation inhibitor Tunicamycin reduces angiogenesis. It also reduces the progression of double negative (ER-/PR-/HER2+) and triple negative (ER-/PR-/HER2-) breast tumor growth in athymic nude mice. Tunicamycin is a competitive inhibitor of N-acetylglucosaminyl 1-phosphate transferase (GPT) activity and down regulates Glc3Man9GlcNAc2-PP-Dol biosynthesis. The consequence is “ER stress“ and the cell cycle arrest in G1. This led us to hypothesize that decreased expression of Wnt protein(s) in Tunicamycin treated endothelial cell would decrease the cell proliferation and angiogenesis. We have used in vitro culture of capillary endothelial cells as model and western blotting, and immunofluorescence microscopy as tools, and observed down regulated expression of Wnt proteins. We, therefore, concluded that Tunicamycin-induced ER stress interferes with the Wnt signaling and inhibits angiogenesis. This study helps us to give explanations on how the Wntsignaling reacts in the presence of Tunicamycin. Also, leads us to think in his possible apoptotic potential.

Cutaneous Microvascular Impairment in 5‐Fluorouracil Treated Cancer Patients

INTRODUCTION Despite frequent use and high efficacy in treatment of several cancers, 5-Fluorouracil (5-FU) chemotherapy is associated with acute and late occurring cardiotoxicity, ranging from vasospastic angina to increased risk of overt cardiovascular disease mortality. To date, the mechanisms by which these toxicities occur are not well understood, with leading theories implicating coronary vascular dysfunction by way of endothelial-dependent vasodilator impairment. PURPOSE & HYPOTHESIS Therefore, the purpose of the present research was to evaluate multiple mechanisms of endothelial function within the cutaneous microcirculation of 5-FU treated cancer patients (5-FU) and matched controls (CON) using the noninvasive measure of laser Doppler flowmetry (LDF). We hypothesized that 5-FU would attenuate both acetylcholine (ACh)-induced and local heating-induced cutaneous vasodilation. METHODS 17 5-FU (13 M, 4 F; age = 63.5 ± 12 yr) and 16 CON (8 M, 8 F; age = 58.1 ± 16 yr) volunteered for this study. Red blood cell flux was acquired via LDF in the forearm at baseline and following 1) iontophoresis of 200 µL of 2% ACh and 2) ~ 35 min of localized heating at 42°C. CVC was calculated from red blood cell flux divided by mean arterial pressure and presented as the percent increase from baseline to peak (%CVC). RESULTS ACh resulted in a significantly increased CVC in both groups, with no differences in %CVC between groups (CON = 920 ± 107%, 5-FU = 842 ± 113%; p = .62). Local heating, however, elicited a significantly lower %CVC in 5-FU treated patients (CON = 567.5 ± 77.7%, 5-FU = 256.7 ± 71.8%; p = .012). CONCLUSION Given the primary role of endothelial nitric oxide in local heating-induced cutaneous vasodilation, but not during ACh, our findings suggest that 5-FU chemotherapy treatment may result in endothelial dysfunction through nitric oxide mediated mechanisms. Experiments to corroborate these findings through iontophoresis of N-ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and in human coronary endothelial cell culture are ongoing by our group and will provide additional mechanistic insight into 5-FU induced cardiotoxicity.

Damage to cardiac vasculature may be associated with breast cancer treatment-induced cardiotoxicity

BackgroundBreast cancer is the most common female cancer worldwide. Effective therapies including doxorubicin and trastuzumab have improved survival, but are associated with a substantial risk of cardiovascular disease. Mechanisms underlying cancer treatment-induced cardiotoxicity (CTC) are poorly understood and have largely focused on cardiomyocyte damage, although other cellular populations in the heart such as the cardiac endothelium, may play an important role in cardiac damage. We treated a breast tumor-bearing mouse model with doxorubicin and trastuzumab to investigate the role of the cardiac endothelium in the development of CTC.MethodsImmune compromised mice were inoculated in the 4th mammary fat pad with human breast cancer cells overexpressing HER2 (BT474). When tumors were palpable, mice were treated weekly with doxorubicin (5 mg/kg) and trastuzumab (4 mg/kg). The cardiac phenotype of mice was assessed by echocardiography and histological evaluation of the heart. Cardiac vascular damage was assayed by in vivo permeability assays and primary cultures of murine cardiac endothelial cells were used to assay doxorubicin toxicity in vitro.ResultsThe growth of BT474 breast tumors in Balb/c Nude mice was suppressed upon treatment with doxorubicin and trastuzumab. Mice treated for 4 months with doxorubicin and trastuzumab maintained body weights, but demonstrated an echocardiographic phenotype consistent with preserved left ventricular (LV) ejection fraction, decreased LV mass and increased filling pressures (E/e’). Histological staining with Masson’s trichrome and Picrosirius red showed extensive fibrosis and increased collagen deposition in the ventricular myocardium surrounding blood vessels of treated mice compared to untreated mice. Evans blue permeability assays demonstrated increased cardiac vasculature permeability while primary cardiac endothelial cells exposed to doxorubicin in vitro showed increased cell death as compared to lung or liver endothelial cells.ConclusionsAn orthotopic mouse model of human breast cancer in Nude mice treated with doxorubicin and trastuzumab resulted in a cardiac vascular defect accompanied by preserved LV ejection fraction, decreased LV mass, suggesting mild diastolic dysfunction and cardiac remodeling consistent with subclinical cardiotoxicity. Our data suggest that cardiac endothelium is more sensitive to doxorubicin therapy as compared to other organ endothelium and cardiac endothelial damage may correlate with breast cancer treatment-induced cardiotoxicity.

Role of microglial and endothelial CD36 in post-ischemic inflammasome activation and interleukin-1β-induced endothelial activation

Cerebral ischemia is associated with an acute inflammatory response that contributes to the resulting injury. The innate immunity receptor CD36, expressed in microglia and endothelium, and the pro-inflammatory cytokine interleukin-1β (IL-1β) are involved in the mechanisms of ischemic injury. Since CD36 has been implicated in activation of the inflammasome, the main source of IL-1β, we investigated whether CD36 mediates brain injury through the inflammasome and IL-1β. We found that active caspase-1, a key inflammasome component, is decreased in microglia of CD36-deficient mice subjected to transient middle cerebral artery occlusion, an effect associated with a reduction in brain IL-1β. Conditional deletion of CD36 either in microglia or endothelium reduced ischemic injury in mice, attesting to the pathogenic involvement of CD36 in both cell types. Application of an ischemic brain extract to primary brain endothelial cell cultures from wild type (WT) mice induced IL-1β-dependent endothelial activation, reflected by increases in the cytokine colony stimulating factor-3, a response markedly attenuated in CD36-deficient endothelia. Similarly, the increase in colony stimulating factor-3 induced by recombinant IL-1β was attenuated in CD36-deficient compared to WT endothelia. We conclude that microglial CD36 is a key determinant of post-ischemic IL-1β production by regulating caspase-1 activity, whereas endothelial CD36 is required for the full expression of the endothelial activation induced by IL-1β. The data identify microglial and endothelial CD36 as critical upstream components of the acute inflammatory response to cerebral ischemia and viable putative therapeutic targets.

Modeling the printability of photocuring and strength adjustable hydrogel bioink during projection-based 3D bioprinting

As a projection based three-dimensional printing method, digital light processing bioprinting (DLPBP) has higher printing resolution and is suitable for constructing finer structures to mimic tissues when compared to extrusion based bioprinting. However, there is a lack of understanding about printing behavior during DLPBP. Herein, a photo crosslinking theory for ink was established and a specified amount of light absorber was added to control crosslinking depth. Then, a standardized methodology was established to quantitatively evaluate printing resolution using different parameters. Complex biostructures, such as the ear, hand, and heart, were precisely printed after understanding the mechanism. Additionally, the mechanical properties of printed samples were accurately adjusted by changing the hydrogel concentration, as well as the degree of substitution and photocrosslinking time. The tissue types printed were from ultra-soft tissues, such as liver (6–8 kPa) to soft tissue, such as the skin (0.3–0.4 MPa). A branching vessel with cells in a real tensile modulus was printed as a demonstration. After 1 week of culture, proliferation and function of human umbilical vein endothelial cells were characterized. Overall, we made it possible to print a mimic complex tissue with high precision, required physical properties and functionalized living cells.

Angiotensin II, ATP and high extracellular potassium induced intracellular calcium responses in primary rat brain endothelial cell cultures.

The meninges shield the nervous system from diverse, rather harmful stimuli and pathogens from the periphery. This tissue is composed of brain endothelial cells (BECs) that express diverse ion channels and chemical-transmitter receptors also expressed by neurons and glial cells to communicate with each other. However, information about the effects of ATP and angiotensin II on BECs is scarce, despite their essential roles in blood physiology. This work investigated in vitro if BECs from the meninges from rat forebrain respond to ATP, angiotensin II and high extracellular potassium, with intracellular calcium mobilizations and its second messenger-associated pathways. We found that in primary BEC cultures, both ATP and angiotensin II produced intracellular calcium responses linked to the activation of inositol trisphosphate receptors and ryanodine receptors, which led to calcium release from intracellular stores. We also used RT-PCR to explore what potassium channel subunits are expressed by primary BEC cultures and freshly isolated meningeal tissue, and which might be linked to the observed effects. We found that BECs mainly expressed the inward rectifier potassium channel subunits Kir1.1, Kir3.3, Kir 4.1 and Kir6.2. This study contributes to the understanding of the functions elicited by ATP and angiotensin II in BECs from rat meninges. SIGNIFICANCE OF THE STUDY: Brain endothelial cells (BECs) express diverse ion channels and membrane receptors, which they might use to communicate with neurons and glia. This work investigated in vitro, if BECs from the rat forebrain respond to angiotensin II and ATP with intracellular calcium mobilizations. We found that these cells did respond to said substances with intracellular calcium mobilizations linked to inositol trisphosphate and ryanodine receptor activation, which led to calcium release from intracellular stores. These findings are important because they might uncover routes of active communication between brain cells and endothelial cells.

Numerical approach-based simulation to predict cerebrovascular shear stress in a blood-brain barrier organ-on-a-chip

Most of the compounds are impermeable to the blood-brain barrier (BBB), which poses a significant challenge in the development of therapeutics for the treatment of neurological diseases. Most of the existing in vitro BBB models are not capable of mimicking the in vivo conditions and functions. The numerical approach-based simulation model was proposed to accurately predict the in vivo level shear stress for the microfluidic BBB-on-a-chip. The in vivo level shear stress was predicted for various conditions of volume flow rates, porosities of the polycarbonate membrane of the BBB model, and dimensions of the microfluidic channel. The in vivo shear stress of the microfluidic BBB model increased with a decrease in the dimension of the microfluidic channel and a decrease in the porosity. The in vivo shear stress predicted by the optimized numerical approach-based simulation was validated within 2.17% error with the experimental in vivo level of shear stress at the porosity of 0.01% and all volume flow rates. The shear stress value, according to the volume flow rate of the microfluidic BBB chip with the optimal microfluidic channel size, was effective for the successful formation of tight junctions in primary endothelial cell culture. In this regard, the proposed method provided a standard for the development of various microfluidic organ-on-chip devices that replicate the in vivo conditions and shear stress.

Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells.

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells.

The Application of a Bone Marrow Mesenchymal Stem Cell Membrane in the Vascularization of a Decellularized Tracheal Scaffold.

Airway stenosis is a common problem in the neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU). A tissue-engineered trachea is a new therapeutic method and a research hotspot. Successful vascularization is the key to the application of a tissue-engineered trachea. However, successful vascularization studies lack a complete description. In this study, it was assumed that rabbit bone marrow mesenchymal stem cells were obtained and induced by ascorbic acid to detect the tissue structure, ultrastructure, and gene expression of the extracellular matrix. A vascular endothelial cell culture medium was added in vitro to induce the vascularization of the stem cell sheet (SCS), and the immunohistochemistry and gene expression of vascular endothelial cell markers were detected. At the same time, vascular growth-related factors were added and detected during SCS construction. After the SCS and decellularized tracheal (DT) were constructed, a tetrandrine allograft was performed to observe its vascularization potential. We established the architecture and identified rabbit bone marrow mesenchymal stem cell membranes by 14 days of ascorbic acid, studied the role of a vascularized membrane in inducing bone marrow mesenchymal stem cells by in vitro ascorbic acid, and assessed the role of combining the stem cell membranes and noncellular tracheal scaffolds in vivo. Fourteen experiments confirmed that cell membranes promote angiogenesis at gene level. The results of 21-day in vitro experiments showed that the composite tissue-engineered trachea had strong angiogenesis. In vivo experiments show that a composite tissue-engineered trachea has strong potential for angiogenesis. It promotes the understanding of diseases of airway stenosis and tissue-engineered tracheal regeneration in newborns and small infants.

Angiogenic Effects of Anxiolytic Fabomotizole.

In vitro experiments performed on an isolated human endothelial HUVEC cell culture showed that the anxiolytic fabomotizole, which, in addition to the anxiolytic effect, has neuroprotective and cardioprotective activities largely associated with its agonistic action on sigma-1 receptors and shows a pronounced angiogenic activity. Fabomotizole angiogenic activity is realized in the range concentration from 10-5 to 10-8 M and is doze-dependent. In the literature, data on the presence of angiogenic activity in sigma receptor agonists have not been previously reported.

Acute Response of Human Aortic Endothelial Cells to Loss of Pulsatility as Seen during Cardiopulmonary Bypass

Cardiopulmonary bypass (CPB) results in short-term (3–5 h) exposure to flow with diminished pulsatility often referred to as “continuous flow”. It is unclear if short-term exposure to continuous flow influences endothelial function, particularly, changes in levels of pro-inflammatory and pro-angiogenic cytokines. In this study, we used the endothelial cell culture model (ECCM) to evaluate if short-term (≤5 h) reduction in pulsatility alters levels of pro-inflammatory/pro-angiogenic cytokine levels. Human aortic endothelial cells (HAECs) cultured within the ECCM provide a simple model to evaluate endothelial cell function in the absence of confounding factors. HAECs were maintained under normal pulsatile flow for 24 h and then subjected to continuous flow (diminished pulsatile pressure and flow) as observed during CPB for 5 h. The ECCM replicated pulsatility and flow morphologies associated with normal hemodynamic status and CPB as seen with clinically used roller pumps. Levels of angiopoietin-2 (ANG-2), vascular endothelial growth factor-A (VEGF-A), and hepatocyte growth factor were lower in the continuous flow group in comparison to the pulsatile flow group whereas the levels of endothelin-1 (ET-1), granulocyte colony stimulating factor, interleukin-8 (IL-8) and placental growth factor were higher in the continuous flow group in comparison to the pulsatile flow group. Immunolabelling of HAECs subjected to continuous flow showed a decrease in expression of ANG-2 and VEGF-A surface receptors, tyrosine protein kinase-2 and Fms-related receptor tyrosine kinase-1, respectively. Given that the 5 h exposure to continuous flow is insufficient for transcriptional regulation, it is likely that pro-inflammatory/pro-angiogenic signaling observed was due to signaling molecules stored in Weible-Palade bodies (ET-1, IL-8, ANG-2) and via HAEC binding/uptake of soluble factors in media. These results suggest that even short-term exposure to continuous flow can potentially activate pro-inflammatory/pro-angiogenic signaling in cultured HAECs and pulsatile flow may be a successful strategy in reducing the undesirable sequalae following continuous flow CPB.