Cultures Of Human Endothelial Cells Research Articles

CLMP Promotes Leukocyte Migration Across Brain Barriers in Multiple Sclerosis.

Background and ObjectivesIn multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor–like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS.MethodsExpression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP.ResultsUsing bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP+ B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP+ immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS.DiscussionCollectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.

The distribution of electrospun polylactic acid in polycaprolactone matrix controlled by traction rate and its effect on the foamed porous tissue engineering scaffolds

AbstractPolycaprolactone (PCL) matrix scaffolds have been extensively studied for tissue engineering applications, which were limited by poor cell adhesion and mechanical strength. Fiber reinforcement modification could be used to improve material properties and optimize material structure. In this study, polylactic acid (PLA) electrospun nanofibers were dispersed in PCL powder, and then the composites were extruded and blended at different traction speed. During the blending and extrusion process, the nanofibrous phase was uniformly dispersed in the matrix, and a kind of tissue engineering scaffold with high connectivity and network structure was prepared using supercritical carbon dioxide foaming. The effect of nanofibers on the foaming behavior of PCL matrix composites prepared at different traction rates was investigated, and the addition of nanofibers promoted the connectivity of the scaffold. Human umbilical vein endothelial cells (HUVECs) culture results showed that the high‐connectivity scaffold was biocompatible and could significantly promote the adhesion and growth of HUVECs. Therefore, fiber‐reinforced porous scaffolds have great application potential in the field of tissue engineering scaffolds.

The Gelatinase Inhibitor ACT-03 Reduces Gliosis in the Rapid Kindling Rat Model of Epilepsy, and Attenuates Inflammation and Loss of Barrier Integrity In Vitro.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of intra- and extracellular proteins. Several brain MMPs have been implicated in neurological disorders including epilepsy. We recently showed that the novel gelatinase inhibitor ACT-03 has disease-modifying effects in models of epilepsy. Here, we studied its effects on neuroinflammation and blood–brain barrier (BBB) integrity. Using the rapid kindling rat model of epilepsy, we examined whether ACT-03 affected astro- and microgliosis in the brain using immunohistochemistry. Cellular and molecular alterations were further studied in vitro using human fetal astrocyte and brain endothelial cell (hCMEC/D3) cultures, with a focus on neuroinflammatory markers as well as on barrier permeability using an endothelial and astrocyte co-culture model. We observed less astro- and microgliosis in the brains of kindled animals treated with ACT-03 compared to control vehicle-treated animals. In vitro, ACT-03 treatment attenuated stimulation-induced mRNA expression of several pro-inflammatory factors in human fetal astrocytes and brain endothelial cells, as well as a loss of barrier integrity in endothelial and astrocyte co-cultures. Since ACT-03 has disease-modifying effects in epilepsy models, possibly via limiting gliosis, inflammation, and barrier integrity loss, it is of interest to further evaluate its effects in a clinical trial.

HIV-1 gp120 and tobacco smoke synergistically disrupt the integrity of the blood-brain barrier

In the United States, the Centers for Disease Control and Prevention (CDC) terms HIV and tobacco use among the ten most important public health challenges we face today. In the last decade, there has been a remarkable decrease in the number of deaths due to HIV/AIDS, especially after the widespread availability and use of combination antiretroviral therapy (cART). However, people living with HIV/AIDS have a heightened risk of chronic complications and comorbidities, including neurological disorders. Around 40–60 % of HIV-infected individuals progress to NeuroAIDS, a group of disorders caused primarily by HIV-mediated damage to the central and peripheral nervous systems, despite receiving cART. The detrimental effects of chronic smoking on the cerebrovascular system are also well studied and reported. Addictive behavior, such as smoking, is more common in HIV patients compared to the general population. In this context, given the existing immune suppression, smoking can pose a significant risk for the progression of the disease to NeuroAIDS by disrupting the integrity of the blood-brain barrier (BBB). Here we show that co-treatment with Tobacco Smoke Extract (TSE) and HIV-1 gp120 (HIV envelope glycoprotein) in primary cultures of human brain microvascular endothelial cells promoted heightened cellular stress responses compared to control and individual treatments. Our findings suggest that a potential synergistic effect between smoke exposure and gp120 can worsen the loss of BBB viability, possibly exacerbating NeuroAIDS progression.

CCL14 exacerbates intraplaque vulnerability by promoting neovascularization in the human carotid plaque

ObjectiveTo examine the role of CCL14 in the neovascularization process and vulnerability progression within carotid plaques by investigating the mechanism of CCL14 regulation of VEGF-A. MethodsWe first performed histological analysis and immunohistochemical staining of human carotid plaque tissue to detect the expression of CCL14, JAK2, STAT3 and VEGF-A. We next examined the protein expression of CCL14, VEGF-A, JAK2, STAT3, and phosphorylation of JAK2 and STAT3 in human carotid atherosclerotic plaques by Western blotting. Finally, we performed in vitro culture of human umbilical vein endothelial cells (HUVEC). In the tube formation assay of HUVEC, we added CCL14 siRNA or VEGF-A siRNA to the culture medium using lentiviral transfection to knock down CCL14 or VEGF-A and grouped them for control assays, and detected the changes in the expression of the above proteins using Western blotting. ResultsHistological and Western blotting analysis of human carotid plaque samples showed that the expression of CCL14 and VEGF-A was higher in the vulnerable plaques than in stable plaques. In the in vitro cultures of HUVEC, CCL14 was found to increase the number and length of intercellularly generated tubular structures. CCL14 increases VEGF-A expression via activating JAK2/STAT3 signaling. ConclusionIn the human carotid plaques, CCL14 promotes angiogenesis by upregulation of VEGF-A via JAK2/STAT3 pathway and thus drives the progression of carotid plaques vulnerability.

Monocytic microparticles enhance proinflammatory cytokine secretion by monocytes and activate endothelial cells

Background: Monocytic microparticles (mMPs) are microparticles derived from activated human monocytes and play an important role in cell-to-cell communication in the circulation. Endothelial cells also exist in the circulation and are thought to have a complex interaction with the released mMPs. Both mMPs and endothelial cells are important players in inflammation. However, the underlying mechanism exerted by mMPs in modulating monocyte and endothelial cell activation during inflammation remains unclear. Methods: Monocytic THP-1 and U937 cells, as well as human blood monocytes, were cultured in the presence or absence of their corresponding mMPs prior to assessing tumor necrosis factor-a (TNF-a), interleukin 1b (IL-1b ), and interleukin 6 (IL-6) secretion. Upon the culture of human umbilical vein endothelial cells (HUVECs) in the presence or absence of mMPs, the expression of CD31, intracellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) by HUVECs and endothelial microparticles (eMPs) was assessed. Results: Our results showed that THP-1 cells and stimulated monocyte-derived mMPs enhanced IL-1b and IL-6 secretion, respectively. Additionally, mMPs derived from stimulated monocytes enhanced the expression of CD31, ICAM-1, and VCAM-1 by HUVECs and increased the release of eMPs. Conclusion: These data suggest that mMPs may exacerbate inflammation through IL-1b and IL-6 activity and facilitate monocyte recruitment to inflammatory sites via CD31, ICAM-1, and VCAM-1.

Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis.

In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm2. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.

Non-triple helical form of type IV collagen alpha1 chain suppresses vascular endothelial-cadherin mediated cell-to-cell junctions.

Non-triple helical collagen polypeptide α1(IV) (NTH α1(IV)) is a gene product of COL4A1 and is secreted as a polypeptide chain without the triple helix structure under physiological conditions. Studies have shown that NTH α1(IV) is up-regulated in and around vascular endothelial cells during neovascularization and vascular-like networks of in vitro angiogenesis models, suggesting its involvement in angiogenesis. In the present study, we examined the effect of NTH α1(IV) on endothelial cell-to-cell junctions, and we found that NTH α1(IV) suppressed VE-cadherin (vascular endothelial cadherin) mediated junctions and promoted cellular migration in human umbilical vein endothelial cell cultures. NTH α1(IV) is potentially a factor that induces VE-cadherin endocytosis and promotes neovascular sprouting and elongation. The possible mechanism entails endocytosis of NTH α1(IV) by its cellular receptor(s), Endo180 and/or other proteins, which results in the clearance of the cellular receptor(s) from the cell surface, thus inducing the endocytosis of VE-cadherin. Because the NC1 domain of the α1 chain of type IV collagen, called arresten, is considered an endogenous inhibitor of angiogenesis, it seems that the single polypeptide chain of NTH α1(IV) has conflicting functions.

Hydroxyapatite/tricalcium phosphate (HA/beta-TCP) scaffold combined with bone and endothelial cells as a potential candidate for oral and maxillofacial bone regeneration.

Cell-based scaffolds have been proposed as useful for tissue repair and bone regeneration in oral and maxillofacial tissue engineering. However, vascularization, a key player in bone regeneration, is often inadequate. To improve this, the use of biphasic calcium phosphate biomaterial (30% hydroxyapatite (HA)-70% tricalcium phosphate (TCP) mixture) HA/beta-TCP-scaffold with defined micron-channel sizes and pore sizes capable of driving angiogenesis, may prove helpful. Therefore, our study aimed to combine the HA/beta-TCP scaffold with in vitro differentiated cells, especially endothelial cells, well-known players in angiogenesis. The HA/beta-TCP-scaffold was combined with primary cultures of human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs). Cells were co-cultured with HA/beta-TCP-scaffold in normoxia (20% oxygen tension) for 14 days. Then, we evaluated: cell adhesion/morphology (Scanning Electron Microscopy and Confocal Microscopy); cell viability (Calcein AM/Propidium Iodide Assay); the presence of multinucleated hOCs (Tartrate Resistant- Acid Phosphatase assay); expression of specific markers of both osteo- and vasculogenic differentiation (Immunohistochemistry). All cell types were viable and well adhered to the entire scaffold surface. Interestingly, bone matrix formation and newly formed vessel-like structures were both detected. Although further investigation is needed, our data proved that differentiated cells combined in vitro with HA/beta-TCP-scaffold prior to implantation could represent a useful strategy for future applications in bone regeneration. Especially, the use of customized micro-vascularized cell-based scaffold would fall within the innovative "personalized medicine” approach.

RGMa Participates in the Blood-Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis.

The infiltration of inflammatory cells into the central nervous system (CNS) through the dysfunctional blood–brain barrier (BBB) was critical in the early stages of MS. However, the mechanisms underlying BBB dysfunction remain unknown. Repulsive guidance molecule-a (RGMa) is involved in the pathogenesis of multiple sclerosis (MS), but its role needs to be further explored. This study aimed to evaluate whether RMGa regulates BBB permeability in endothelial cells and MS, and if so, what mechanism may be involved. We created an experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice and a human brain microvascular endothelial cell (HBMEC) culture. The permeability of the BBB is measured in response to various interventions. Our results showed that RGMa is expressed in the endothelial cells in HBMECs and EAE mice. RGMa and its signaling counterpart, bone morphogenetic protein 2 (BMP2)/bone morphogenetic protein receptor type II (BMPRII), were gradually increased as the disease progressed. Moreover, as EAE progressed and the BBB was disrupted, the downstream effector, yes-associated protein (YAP), as well as the tight junctional proteins zonula occludens 1 (ZO-1) and claudin-5, decreased significantly. The permeability assay revealed that lentivirus-induced RGMa overexpression in HBMECs caused a significant breakdown of the BBB, whereas RGMa knockdown significantly strengthens the integrity of the BBB. Furthermore, specifically activating BMPR II or inhibiting YAP based on RGMa knockdown results in a significant decrease of ZO-1 and claudin-5 in vitro. On the contrary, inhibition of BMPR II or activation of YAP after upregulating RGMa prevents the downregulation of ZO-1 and claudin-5 in HBMECs. In addition, serum-soluble RGMa (sRGMa) levels were significantly higher in MS patients, particularly in MS patients with Gd+ lesions, indicating that the BBB has been disrupted. In conclusion, this study shows that RGMa causes BBB dysfunction in endothelial cells via BMP2/BMPR II/YAP, resulting in BBB integrity disruption in MS and that it could be a novel therapeutic target for BBB permeability in MS.

Resolvin E1 attenuates doxorubicin-induced endothelial senescence by modulating NLRP3 inflammasome activation

Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10–100 nM) increased the number of SA-β-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1β. In fact, IL-1β itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1β by interfering with the increased expression of pP65, NLRP3, and pro-IL-1β proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1β axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.

Genetic deletion of endothelial Caveolin‐1 is protective against metabolic disease

In metabolic disease, the endothelium is exposed to a substantial increase in fatty acid concentrations creating a pathological role for endothelial cells (ECs) as lipid metabolizing cells. One mechanism whereby lipids may enter ECs is by caveolin‐1 (Cav1)‐mediated endocytosis. Because both ECs and adipocytes both have abundant Cav1 and both can readily take up lipids, we wanted to differentiate the role of Cav1 between the two cell types. For these reasons, we used an inducible EC‐specific Cav1 knockout mouse (Cav1fl/fl/Cdh5‐CreERT2+). Using these mice, we induced Cav1 genetic deletion from endothelium at 6 weeks, and began a high fat diet (HFD; 60% fat) to induce metabolic disease at 8 weeks. Normal chow fed littermates were used as controls. All mice were examined at 20 weeks (i.e., 12 weeks of HFD). Thus, we had four cohorts: Cre+ HFD, Cre‐ HFD, Cre+ normal chow, and Cre‐ normal chow all injected with tamoxifen. All mice regardless of genotype had comparable weight gain within diet groups, and gonadal fat pad mass. Food consumption was similar between groups, and electron microscopy was used to confirm presence of caveola in adipocytes, but not endothelium. Initial experiments examining plasma lipids found HFD Cre+ mice had both increased serum cholesterol (175mg/dL) and LDL (75mg/dL) compared to HFD Cre‐ controls (cholesterol, 125mg/dL; LDL, 60mg/dL). When these mice were challenged with a bolus of insulin, the blood glucose of Cre+ HFD mice dropped significantly resembling an insulin tolerance curve from normal chow fed controls. Similarly, Cre+ HFD mice were protected from hyperglycemia after bolus glucose injections, resulting in a glucose tolerance phenotype similar to that of a normal chow fed mouse (HFD Cre+ AUC 3000 vs. HFD Cre‐ AUC 60000). There was no difference in Cre+ or Cre‐ mice on normal chow diets with serum lipids or after challenge with insulin or glucose. It isn’t clear why the insulin and glucose protection correspond with significantly increased lipids in the Cre+ HFD mice. For these reasons, we began investigating the possible mechanisms with primary cultures of Human Adipose Microvascular Endothelial Cells (HAMECs) and knocked down Cav1 with siRNA. In monolayers of HAMEC without any other cell type present, we did not observe altered lipid uptake after addition of 50µM of exogenous fatty acids with a 70% knockdown of Cav1 protein. Additionally, HAMECs seahorse assays were used to determine if there were changes in metabolic function when Cav1 was knocked down. This work provides initial evidence for the unique metabolic roles of endothelial Cav1 during periods of high lipid circulation.

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior.

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.

Topical Nanoemulsion of a Runt-related Transcription Factor 1 Inhibitor for the Treatment of Pathologic Ocular Angiogenesis.

Purpose:To test the efficacy of runt-related transcription factor 1 (RUNX1) inhibition with topical nanoemulsion containing Ro5-3335 (eNano-Ro5) in experimental ocular neovascularization.Design:Preclinical experimental study.Participants:In vitro primary culture human retinal endothelial cell (HREC) culture. C57BL/6J 6- to 10-week-old male and female mice.Methods:We evaluated the effect of eNano-Ro5 in cell proliferation, cell toxicity, and migration of HRECs. We used an alkali burn model of corneal neovascularization and a laser-induced model of choroidal neovascularization to test in vivo efficacy of eNano-Ro5 in pathologic angiogenesis in mice. We used mass spectrometry to measure penetration of Ro5-3335 released from the nanoemulsion in ocular tissues.Main Outcome Measures:Neovascular area.Results:RUNX1 inhibition reduced cell proliferation and migration in vitro. Mass spectrometry analysis revealed detectable levels of the active RUNX1 small-molecule inhibitor Ro5-3335 in the anterior and posterior segment of the mice eyes. Topical treatment with eNano-Ro5 significantly reduced corneal neovascularization and improved corneal wound healing after alkali burn. Choroidal neovascularization lesion size and leakage were significantly reduced after treatment with topical eNano-Ro5.Conclusions:Topical treatment with eNano-Ro5 is an effective and viable platform to deliver a small-molecule RUNX1 inhibitor. This route of administration offers advantages that could improve the management and outcomes of these sight-threatening conditions. Topical noninvasive delivery of RUNX1 inhibitor could be beneficial for many patients with pathologic ocular neovascularization.

Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification

Currently, most in vitro engineered bone tissues do not contain viable blood vessel systems, so the vascularization depends on post-implantation angiogenesis from the host, which is often insufficient for repairing large bone defects. In this study, we aimed to create pre-vascularized bone-like tissue from human bone marrow-derived mesenchymal stem cells (HBMSCs) within the self-generated extracellular matrix by simulating the developmental endochondral ossification. Afterward, a three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs)/HBMSCs was introduced to cover bone-like constructs surface for vascularization. Lastly, the pre-vascularized bone-like tissues were subcutaneously implanted into mice and the quality of newly formed blood vessels and bones were later assessed. We particularly examined whether the pre-existing HUVECs/HBMSCs vascular networks within the implants were able to integrate with the host’s blood vessels and facilitate bone formation. Our results showed that this developmentally informed procedure resulted in a robust osteogenic differentiation of HBMSCs. Moreover, the bone-like constructs markedly promoted HUVEC/HBMSCs network formation in vitro. After 28 days of implantation in mice, the experimental group, in which bone-like constructs were pre-vascularized with HUVEC/HBMSCs networks, exhibited significantly more functional blood vessels than the control group that contained HUVEC and HBMSC single cells. Interestingly, increased levels of bone formation and absorption markers were also observed in the pre-vascularized bone-like constructs. Taken together, these findings demonstrated the potential of pre-vascularized bone-like constructs in repairing bone defects.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg−1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

Uremic Toxin Lanthionine Induces Endothelial Cell Mineralization In Vitro.

Vascular calcification (VC) is a pathological event caused by the unusual deposition of minerals in the vascular system, representing the leading cause of cardiovascular mortality in chronic kidney disease (CKD). In CKD, the deregulation of calcium and phosphate metabolism, along with the effect of several uremic toxins, act as key processes conveying altered mineralization. In this work, we tested the ability of lanthionine, a novel uremic toxin, to promote calcification in human endothelial cell cultures (Ea.hy926). We evaluated the effects of lanthionine, at a concentration similar to that actually detected in CKD patients, alone and under pro-calcifying culture conditions using calcium and phosphate. In pro-calcific culture conditions, lanthionine increased both the intracellular and extracellular calcium content and induced the expression of Bone Morphogenetic Protein 2 (BMP2) and RUNX Family Transcription Factor 2 (RUNX2). Lanthionine treatment, in pro-calcifying conditions, raised levels of tissue-nonspecific alkaline phosphatase (ALPL), whose expression also overlapped with Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1) gene expression, suggesting a possible role of the latter gene in the activation of ALPL. In addition, treatment with lanthionine alone or in combination with calcium and phosphate reduced Inorganic Pyrophosphate Transport Regulator (ANKH) gene expression, a protective factor toward the mineralizing process. Moreover, lanthionine in a pro-calcifying condition induced the activation of ERK1/2, which is not associated with an increase in DKK1 protein levels. Our data underscored a link between mineral disease and the alterations of sulfur amino acid metabolisms at a cell and molecular level. These results set the basis for the understanding of the link between uremic toxins and mineral-bone disorder during CKD progression.

Adult Human Vascular Smooth Muscle Cells on 3D Silk Fibroin Nonwovens Release Exosomes Enriched in Angiogenic and Growth-Promoting Factors.

Background. Our earlier works showed the quick vascularization of mouse skin grafted Bombyx mori 3D silk fibroin nonwoven scaffolds (3D-SFnws) and the release of exosomes enriched in angiogenic/growth factors (AGFs) from in vitro 3D-SFnws-stuck human dermal fibroblasts (HDFs). Here, we explored whether coronary artery adult human smooth muscle cells (AHSMCs) also release AGFs-enriched exosomes when cultured on 3D-SFnws in vitro. Methods. Media with exosome-depleted FBS served for AHSMCs and human endothelial cells (HECs) cultures on 3D-SFnws or polystyrene. Biochemical methods and double-antibody arrays assessed cell growth, metabolism, and intracellular TGF-β and NF-κB signalling pathways activation. AGFs conveyed by CD9+/CD81+ exosomes released from AHSMCs were double-antibody array analysed and their angiogenic power evaluated on HECs in vitro. Results. AHSMCs grew and consumed D-glucose more intensely and showed a stronger phosphorylation/activation of TAK-1, SMAD-1/-2/-4/-5, ATF-2, c-JUN, ATM, CREB, and an IκBα phosphorylation/inactivation on SFnws vs. polystyrene, consistent overall with a proliferative/secretory phenotype. SFnws-stuck AHSMCs also released exosomes richer in IL-1α/-2/-4/-6/-8; bFGF; GM-CSF; and GRO-α/-β/-γ, which strongly stimulated HECs’ growth, migration, and tubes/nodes assembly in vitro. Conclusions. Altogether, the intensified AGFs exosomal release from 3D-SFnws-attached AHSMCs and HDFs could advance grafts’ colonization, vascularization, and take in vivo—noteworthy assets for prospective clinical applications.

Spatially arranged encapsulation of stem cell spheroids within hydrogels for the regulation of spheroid fusion and cell migration

Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity than individual cells in suspension. However, there is limited information on the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in a hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into a Matrigel for the investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in a Matrigel, denoted as sandwich culture, with a specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we performed a sandwich culture of human umbilical vein endothelial cells (HUVECs) and ASCs in co-cultured spheroids in the Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when the inter-spheroid distance was 200 µm. Similar results were obtained from an in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in a hydrogel. Statement of significanceRecently, spheroid-based 3D tissue culture techniques such as spheroid encapsulation or 3D printing are being intensively investigated for various purposes. However, there is limited research regarding the effect of the inter-spheroid distance on spheroid communication. Here, we demonstrate a spatially arranged spheroid encapsulation method within a Matrigel by using a temperature-responsive hydrogel. Human adipose-derived stem cell spheroids are encapsulated with a precisely controlled inter-spheroid distance from 100 to 400 µm and show different tendencies in cell migration and spheroid fusion. Our results suggest that the inter-spheroid distance affects spheroid communication, and thus, the inter-spheroid distance needs to be considered carefully according to the purpose.

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.