Cultures Of Endothelial Cells Research Articles

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line

BackgroundBlood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.MethodsThe model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).ResultsThe resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane.ConclusionsIn conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Development of a 3D Perfused In Vitro System to Assess Proangiogenic Properties of Compounds.

Perturbation of angiogenesis is associated with a variety of diseases and pro- as well as antiangiogenic therapies are being actively explored. Additionally, unintended adverse drug effects on angiogenesis might lead to promotion of tumor progression and cardiovascular complications. Several tri-dimensional microfluidic vessel-on-chip systems have been described that allow a more accurate investigation of vascular physiology and pathology, compared to the two-dimensional static culture of endothelial cells. The OrganoPlate® angiogenesis-on-chip system has been demonstrated to be amenable to high-throughput screening for the antiangiogenic properties of molecules. We set out to adapt this system for high-throughput screening of molecules with proangiogenic properties. Our technical advancement of the OrganoPlate® angiogenesis-on-chip assay expands its applicability in the early screening of both anti- as well as proangiogenic properties of compounds for therapeutic modulation of angiogenesis as well as the identification of angiogenesis-associated drug-induced vascular toxicities.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that exvivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Exvivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Flow-dependent regulation of rat mesenteric lymphatic vessel contractile response requires activation of endothelial TRPV4 channels.

The objective of our study is to evaluate the involvement of the transient receptor potential vanilloid 4 (TRPV4) in the alteration of lymphatic pumping in response to flow and determine the signaling pathways involved. We used immunofluorescence imaging and western blotting to assess TRPV4 expression in rat mesenteric lymphatic vessels. We examined inhibition of TRPV4 with HC067047, nitric oxide synthase (NOS) with L-NNA and cyclooxygenases (COXs) with indomethacin on the contractile response of pressurized lymphatic vessels to flow changes induced by a stepwise increase in pressure gradients, and the functionality of endothelial TRPV4 channels by measuring the intracellular Ca2+ response of primary lymphatic endothelial cell cultures to the selective agonist GSK1016790A. TRPV4 protein was expressed in both the endothelial and the smooth muscle layer of rat mesenteric lymphatics with high endothelial expression around the valve sites. When maintained under constant transmural pressure, most lymphatic vessels displayed a decrease in contraction frequency under conditions of flow and this effect was ablated through inhibition of NOS, COX or TRPV4. Our findings demonstrate a critical role for TRPV4 in the decrease in contraction frequency induced in lymphatic vessels by increases in flow rate via the production and action of nitric oxide and dilatory prostanoids.

Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve With Raphe Formation.

A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for BAV treatment. Over the past decade, the genes involved in aortic valve development and BAV formation have been increasingly recognized. On the other hand, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were performed to evaluate the expression pattern in the aortic valve. Accordingly, related genetic mouse models (both knockout and knockin) were generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method to further study the roles of ADAMTS family genes. The lineage-tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Inducible pluripotent stem cells derived from both BAV patients and genetic mouse tissue were used to study the molecular mechanism of ADAMTS. Immunohistochemistry was performed to examine the phenotype of cardiac valve anomalies, especially in the extracellular matrix components. ADAMTS genes targeting and phenotype screening in zebrafish and targeted DNA sequencing on a cohort of patients with BAV identified ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs 16) as a BAV-causing gene and found the ADAMTS16 p. H357Q variant in an inherited BAV family. Both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited a right coronary cusp-noncoronary cusp fusion-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Further, ADAMTS16 deficiency in Tie2 lineage cells recapitulated the BAV phenotype. This was confirmed in lineage-tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using inducible pluripotent stem cells-derived endothelial cells and genetic mouse embryonic heart tissue unveiled enhanced FAK (focal adhesion kinase) signaling, which was accompanied by elevated fibronectin levels. Both in vitro inducible pluripotent stem cells-derived endothelial cells culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

Preparation and evaluation of a suspension of human corneal endothelial cells isolated from the eyes of cadaveric donors for transplantation in an <em>ex vivo</em> experiment

Background. According to the World Health Organization, corneal diseases are one of the major causes of blindness globally. Endothelial dystrophy is one of the etiological factors leading to corneal diseases. The corneal endothelium is a monolayer of cells with virtually no mitotic activity. When the density of corneal endothelial cells falls below a critical threshold, the endothelium loses its ability to regulate corneal stromal hydration. This leads to corneal clouding and, consequently, to reduced visual acuity and quality of life of the patient. In this regard, various keratoplasty methods are widely used in clinical practice. Today, it is technically possible to transplant all corneal layers via penetrating keratoplasty, and to transplant the posterior epithelium via layer-bylayer keratoplasty. These surgical approaches are now widely used in everyday practice, but they require the use of scarce material – cadaveric donor corneas, from which grafts for the above-mentioned operations are formed in the conditions of an eye bank. In this regard, protocols for obtaining human corneal endothelial cell (HCEC) culture for subsequent transplantation have been proposed in recent years. However, the use of such approaches in Russia is limited by the law. The aim of this study was to experimentally justify the possibility of transplanting uncultured endothelial cells, isolated from cadaveric human corneas. Materials and methods. The first stage of the work consisted of obtaining a suspension of endothelial cells from cadaveric donor corneas and studying it; at the second stage, the transplantation effectiveness of the resulting cell suspension was assessed in an ex vivo experiment. Results. The cell phenotype after transplantation by the proposed method had high viability and preservation. Conclusions. The presented results suggest that phenotype and adhesion ability are preserved, and that the cell suspension has a high level of viability under adequate loss of endothelial cells during transplantation in the ex vivo experiment.

Human calcified aortic valves release procoagulant microparticles inducing endothelial dysfunction and thrombogenicity: Role of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Background Aortic stenosis (AS) pathogenesis is a dynamic process with interplay between the hemostatic system and the valve leading to inflammation, oxidative stress, endothelial dysfunction, and calcification. Within the pathological valve, mechanical strain induces the release of cell-derived microparticles (MPs) that promote mineralization and carry a pro-oxidant activator signal. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) showed cardiovascular benefit and anti-inflammatory responses in patients with heart failure. Oxidative stress has been shown to trigger SGLT2 expression in endothelial cells (ECs) to induce endothelial dysfunction. Therefore, this study evaluated calcified AS valves and the effect of AS-derived MPs on ECs and, if so, the role of the SGLT2. Methods Pathological valves were collected from 90 AS and 15 aortic insufficiency (AI) patients. MPs were extracted from human pathological valves, and quantified through their prothrombinase activity. Primary cultures of porcine valvular endothelial cells (VECs) were treated with MPs (10 nM) or thrombin (1 U/ml). Protein expression levels were assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-qPCR, oxidative stress using dihydroethidium, and nitric oxide (NO) generation by DAF-FM diacetate. THP1 cells were used to evaluate the adhesiveness of VECs, and the generation of thrombin using a specific chromogenic substrate. Results Calcified AS valves showed increased levels of thrombogenic (TF, PAI-1), adhesive (VCAM-1, ICAM-1) and inflammatory markers (CD68, p-p65 NF-κB) and SGLT2 compared to non-calcified valves. These responses were associated with a higher level of oxidative stress that was reduced by inhibitors of the angiotensin system (ACEi, AT1R) and SGLT2, and an increased release of procoagulant MPs. Exposure of VECs to AS-MPs increased the expression of markers of the angiotensin system (ACE, p67 phox), inflammation (CXCL10, CCL11, CXCL8), thrombogenicity (TF, PAI-1), angiogenesis (VEGFR2, ANGPTL4), matrix remodeling (BMP-2, MMP-1), and SGLT2 and caused the down-regulation of eNOS. These responses were associated with a sustained pro-oxidant response, the nuclear translocation of NF-kB and blunted bradykinin-induced NO formation, which were prevented by inhibition of AT1R, NADPH oxidases, SGLT2, and NF-kB. AS-MPs-treated VECs showed also increased adhesion of THP-1 cells, and generation of thrombin. Conclusions Calcified AS valve is a potent reservoir of MPs, acting as a pro-thrombogenic source per se and prompting valvular endothelial cells dysfunction through activation of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. The observed protective effect of empagliflozin suggests SGLT2 inhibitors as a novel therapeutic option to curb valve dysfunction.

Development of a human in vitro model of bone marrow vascular niche on a chip to study CD34+ hematopoietic stem/progenitor cells function in diabetes

Abstract Background/Introduction Protective bone marrow (BM) CD34+ hematopoietic stem/progenitor cells (HSPCs) are released in the bloodstream after cardiomacrovascular (CV) tissue damage such as myocardial infarction1. Diabetes, an high risk condition to develop CV diseases, results in BM damage2-4 and detrimental pro-apoptotic and pro-oxidant shifts in BM-derived CD34+HSPCs5,6. Being able to classify and study the mechanisms of CD34+HSPC-vascular cell interaction would be a great step forward in the CV research field. However, nowadays there is no available primary human cell-based in vitro model to study the BM vascular niche. Purpose We aim to develop a human-based in vitro model to reproduce the BM vascular niche that allows the analysis of molecular mechanisms involved in CD34+HSPC function. This model will be relevant to identify molecular targets beyond hypoglycemic treatment and potentiate personalized medicine applications for diabetic complications. Method Human endothelial cells and pericytes were isolated from femoral head of patient who underwent hip replacement or from ad hoc BM aspirate by magnetic bead separation and characterized by immunofluorescence staining and flow cytometry. A culture of endothelial and pericytic cells to replicate the BM vascular niche was developed on a chip. CD34+HSPC subpopulation alteration in diabetes was preliminarily evaluated through single cell sequencing analysis (scRNAseq) on CD34+HSPC cells isolated from BM of patients with or without diabetes (N=1). Results We successfully set up a new method to efficiently isolate autologous BM endothelial cells and pericytes derived from patients (N=3). The cells expressing typically endothelial (CD31, ZO-1, and VE-cadherin) and pericytic (CD146, CD140b) markers were cultured in a 2D chip model. ScRNAseq reveals 15 clusters of CD34+HSPCs and important differences between the non-diabetic and diabetic samples. In particular, cluster 2 is highly represented in the non-diabetic and completely disappears in the diabetic sample, while on the contrary cluster 9 is over-represented in the diabetic compared to the non-diabetic sample. Interestingly, though the trajectory analysis cluster 2 shows the highest probability of turning into cluster 9. Through Ingenuity Canonical Pathways analysis we identified the anti-oxidant pathway as the most affected in diabetes-derived CD34+HSPCs. Conclusions In vitro human vascular niche model could be the gold standard to study BM cell crosstalk. In our interest, cluster 2 completely disappears in diabetes-derived CD34+HSPCs, potentially representing the CV-protective CD34+HSPC fractions more prone to be mobilized. Developing of our in vitro model would be useful to validate this hypothesis.Figure 1:scRNAseq cluster UMAPFigure 2:Endothelial cells IF staining

An effective method for culturing functional human corneal endothelial cells using a xenogeneic free culture medium

Endothelial dysfunction is a leading cause of corneal blindness in developed countries and the only available treatment is the endothelial transplantation. However, the limited availability of suitable donors remains a significant challenge, driving the exploration of alternative regenerative therapies. Advanced Therapy Medicinal Products show promise but must adhere to strict regulations that prohibit the use of animal-derived substances. This study investigates a novel culture methodology using Plasma Rich in Growth Factors (PRGF) as the only source of growth factors for primary cultures of human corneal endothelial cells (CECs). CECs were obtained from discarded corneas or endothelial rings and cultured in two different media: one supplemented with xenogeneic factors and other xenogeneic-free, using PRGF. Comprehensive characterization through immunofluorescence, morphological analyses, trans-endothelial electrical resistance measurements, RNA-seq, and qPCR was conducted on the two groups. Results demonstrate that CECs cultured in the xenogeneic-free medium exhibit comparable gene expression, morphology, and functionality to those cultured in the xenogeneic medium. Notably, PRGF-expanded CECs share 46.9% of the gene expression profile with native endothelium and express all studied endothelial markers. In conclusion, PRGF provides an effective source of xenogeneic-free growth factors for the culture of CECs from discarded corneal tissue. Further studies will be necessary to demonstrate the applicability of these cultures to cell therapies that make clinical translation possible.

RNA surveillance by ADAR1 is required for vascular endothelial resilience

Abstract Introduction Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive. Methods Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects. Results EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings. Conclusion Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.

Abstract 18731: Connexin 43 Hemichannels Contribution to Ischemia-Reperfusion-Induced Hyperpermeability

Introduction: Ischemia-reperfusion (I-R) injury is a significant complication in vascular surgery or organ transplantation. I-R injury involves intracellular Ca2+ overload, nitric oxide (NO), oxidative stress. I-R injury increases endothelial vascular hyperpermeability leading to tissue edema and damage mediated by endothelial Ca2+ increase. The role of Ca2+ permeable channels in I-R is not defined. Connexin (Cx) proteins form hemichannels, allowing the exchange of molecules between intracellular and extracellular spaces. The Cx43 isoform is highly expressed in endothelial cells (EC). Endothelial Ca2+ increase promotes NO-dependent Cx43 S-nitrosylation. However, whether Cx43 hemichannels are involved in I-R-induced hyperpermeability is unknown. Hypothesis: VEGF-induced hyperpermeability causes increased NO-mediated Cx43 S-nitrosylation and opening of endothelial Cx43 hemichannels, leading to Ca2+ entry and exacerbating reperfusion damage. Methods: We used VEGF as I-R related pro-inflammatory agonist. Using primary cultures of mesenteric endothelial cells, we tested Cx43 hemichannel activity by ethidium uptake and Ca2+ influx with Fluo-4 indicator. We evaluated hyperpermeability in vitro and in vivo Results: Results indicate that VEGF stimulation increased hemichannel activity in EC, associated with a rapid rise of Ca2+ influx. Both hemichannel activation and Ca2+ increase were abolished by treatment with specific Cx43 blocker (Gap19) or blocking NO production with NG-monomethyl-L-arginine (LNMMA). Cx43 inhibition blunted VEGF-induced hyperpermeability both in vitro in primary EC culture and in vivo mouse mesenteric vascular bed. To further understand the participation of NO-mediated S-nitrosylation in the VEGF-induced hyperpermeability, we replaced cysteine 271 by serine to create a Cx43C271S mouse that fails to cause S-nitrosylation of Cx43. We demonstrated that this knockin mouse exhibits a decrease in VEGF-stimulated hyperpermeability in vivo. Conclusions: These results indicate that Cx43 hemichannels activation is critical to VEGF (I-R inflammatory environment)-induced hyperpermeability and the S-nitrosylated Cx43 is a key regulator in I-R damage. AHA support: # 932684 and #23DIVSUP1054931

Abstract 18413: Therapeutic Genome Editing of Endothelial FABP4 by Endothelium-Targeted Nanoparticle Delivery of the CRISPR/Cas9 System Inhibits Pulmonary Arterial Hypertension in Mice and Rats

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling. The molecular mechanisms of obliterative pulmonary vascular remodeling remain elusive. Hypothesis: Therapeutic genome editing of endothelial Fabp4 will knockout FABP4 expression selectively in endothelial cells and inhibit PAH in mice and rats. Methods: Employing the newly developed endothelial cell (EC)-targeted nanoparticle delivery technology, plasmid DNA expressing CRISPR/Cas9 controlled by Cdh5 promoter and Fabp4 -specific guide RNAs driven by U6 promoter was administered i.v. to adult SD rats and Egln1 Tie2Cre mice which exhibit severe PAH and obliterative pulmonary vascular remodeling. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, right ventricular systolic pressure (RVSP), ratio of right ventricular versus left ventricular plus septum (RV/LV+S) and pulmonary vascular remodeling were determined. Pharmacological FABP4 inhibitor was also employed to treat MCT rats as a comparison. Similarly, the pulmonary hypertensive phenotype in genome-edited Egln1 Tie2Cre mice was also characterized. Results: Nanoparticle delivery of the all-in-one CRISPR rCdh5 plasmid DNA knockout Fabp4 selectively in endothelial cells in adult rats and Egln1 Tie2Cre mice leading to reduced RVSP, RV hypertrophy and pulmonary vascular remodeling in MCT rats and also in Egln1 Tie2Cre mice. Obliterative pulmonary vascular remodeling seen in Egln1 Tie2Cre mice was inhibited and the mortality rate in endothelial Fabp4-deficient Egln1 Tie2Cre mice was reduced. Treatment of FABP4 inhibitor also inhibits PAH in MCT rats. We also observed a marked increase of FABP4 expression in endothelial cells of lung samples of IPAH patients. Overexpression of FABP4 in primary cultures of human lung microvascular endothelial cells resulted in a 70% increase of cell proliferation. Conclusions: These findings demonstrate endothelial FABP4 is a promising target for PAH therapy. EC-targeted nanoparticle delivery of the genome editing system may represent an effective and safer gene therapy approach for PAH treatment.

Mechanosignaling-related angiocrine factors drive osteoblastic phenotype in response to zirconia

BackgroundThe growing use of zirconia as a ceramic material in dentistry is attributed to its biocompatibility, mechanical properties, esthetic appearance, and reduced bacterial adhesion. These favorable properties make ceramic materials a viable alternative to commonly used titanium alloys. Mimicking the physiological properties of blood flow, particularly the mechanosignaling in endothelial cells (ECs), is crucial for enhancing our understanding of their role in the response to zirconia exposure. MethodsIn this study, EC cultures were subjected to shear stress while being exposed to zirconia for up to 3 days. The conditioned medium obtained from these cultures was then used to expose osteoblasts for a duration of 7 days. To investigate the effects of zirconia on osteoblasts, we examined the expression of genes associated with osteoblast differentiation, including Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Additionally, we assessed the impact of mechanosignaling-related angiocrine factors on extracellular matrix (ECM) remodeling by measuring the activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) during the acquisition of the osteogenic phenotype, which precedes mineralization. ResultsOur data revealed that mechanosignaling-related angiocrine factors play a crucial role in promoting an osteoblastic phenotype in response to zirconia exposure. Specifically, exposed osteoblasts exhibited significantly higher expression levels of genes associated with osteoblast differentiation, such as Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Furthermore, the activities of MMP2 and MMP9, which are involved in ECM remodeling, were modulated by mechanosignaling-related angiocrine factors. This modulation is likely an initial event preceding the mineralization phase. ConclusionBased on our findings, we propose that mechanosignaling drives the release of angiocrine factors capable of modulating the osteogenic phenotype at the biointerface with zirconia. This process creates a microenvironment that promotes wound healing and osseointegration. Moreover, these results highlight the importance of considering the mechanosignaling of endothelial cells in the modulation of bone healing and osseointegration in the context of blood vessel effects. Our data provide new insights and open avenues for further investigation into the influence of mechanosignaling on bone healing and the osseointegration of dental devices.

Radchip: A Laminin-Lined Red Blood Cell (RBC) Adhesion Device for Functional Characterization and Clinical Evaluation of Sickle RBCs at Steady-State

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the beta-globin gene producing an abnormal hemoglobin (HbS) which, under deoxygenation, polymerizes and causes severe distortion of the red blood cell (RBC). RBCs may be dehydrated, poorly deformable and highly adhesive to the endothelium, with abnormal adhesion being of primary interest to this study. Recently, several new disease modifying drugs have been FDA approved, with more in development. These novel agents target specific aspects of SCD pathophysiology, including adhesion. Changes induced by such therapies are not always detectable by common conventional lab instruments. LORRCA (oxygen-gradient ektacytometry) is the gold-standard for deformability measurement; it has been well described both analytically and clinically in SCD. Adhesion and deformability have a dynamic, yet complementary relationship that we explore here. While these may behave in an interdependent manner, each determinant must be considered independently. There is a growing demand for innovative tools to evaluate rheological properties under physiological conditions, particularly microfluidic devices. However, many current devices are limited in their clinical utility due to, choice of ligand, complexity of design, or reliance on endothelial cell culture (typically HUVEC cells) which are difficult to use, vary with passage number, and do not represent adult SCD endothelium. We developed a Red blood cell Adhesion Chip (RAdChip) to model RBC to endothelial adhesion using laminin, a physiologically relevant ligand, to easily, effectively, and reproducibly measure RBC adhesion. Here, we describe our analytic and clinical validation of our device. Peripheral blood samples were collected in EDTA from individuals with SCD and healthy controls under an IRB approved protocol. RBCs were extracted via centrifugation, washed and resuspended in PBS to a 0.5% HCT. Microfluidic devices were fabricated using polydimethylsiloxane (PDMS). A 5ug/ml solution of recombinant laminin was applied to each channel, then blocked with 1% bovine serum albumin (BSA). Resuspended RBCs were introduced to the device through tubing connected to the inlet port. Harvard Apparatus syringe pumps were used to perfuse samples and wash channels at ~1dyne/cm 2. Adhesion was visualized with a Keyence inverted bright-field microscope at 20x (Itasca, IL). Adhered cells were counted within a 1.8mm 2 region of interest (Roi) using the iCLOTS automated cell quantification software (Fay et al, Nat comm accepted 7/2023). We measured adhesion index via RAdChip for common SCD genotypes: HbSS/SB0: (median (x͂) =756; range (r): 154-1889; n=26) HbSC/SB+ : (x͂ =299; r=: 114-731; n=11 ) HbAA: (x͂) =13; r= 5-90; n=7). RBC adhesion values were independent of age (p=0.65) and gender (p=0.63). An increase of 0.1 unit in EImin and EImax decreases RBC adhesion by 214 and 268 cells/mm 2, respectively. A 1mmHg increase in PoS increased adhesion by 19.5 cells/mm 2. sRBC adhesion had a significant positive association with vaso-occlusive events (VOE) (p=0.005), absolute neutrophil count (ANC) (p=0.005), and EImax (p<0.001). Notably, analytical validation of RAdChip produced a mean CV of 0.13 ± 0.94. RAdChip adds a key component of functional RBC evaluation that conventional lab equipment does not measure. We have analytically validated our device, demonstrating an excellent coefficient of variability. 44 patient samples, each examined in triplicate produced a mean CV of 0.13. We have demonstrated clinical validity of our device by establishing correlations with known markers of severity and pain event frequency. RBC adhesion values increase as the number of VOE events increase, reaffirming the belief that adhesion is a principal element in the multistep VOE cascade. In like manner, improvements in deformability are associated with decreased RBC adhesion, strengthening the interdependent nature of VOE molecular events. Recognizing the dynamic nature of SCD, it is necessary to explore sickle erythrocyte adhesion longitudinally to investigate changes before, during, and after VOE occurrences. Continued and increased integration of adhesion measurements with RAdChip and other rheology devices into routine SCD care may be helpful in assessing disease severity, identifying need for additional disease modifying agents, and assist with second line agent selection and monitoring.

NOX1 modulates proliferation, while eNOS regulates migration in the wound healing of corneal endothelial cells in culture

NOX1 modulates proliferation, while eNOS regulates migration in the wound healing of corneal endothelial cells in culture

Inhibitory Effect of Heparin/Polyarginine/Hyaluronic Acid Complex on Coronary Artery Restenosis by Loading Vascular Endothelial Growth Factor

This research was aimed to investigate the inhibitory effect of heparin/polyarginine/hyaluronic acid (HPHA) complex loaded with vascular endothelial growth factor (VEGF) on coronary artery restenosis (CAR). Dopamine was grafted to obtain dopamine-hyaluronic acid (DPA) composite material, and heparin/polyarginine (HP) nanoparticles (NPs) loaded with VEGF were obtained through electrostatic binding (ESB). The HP NPs loaded with VEGF mixed with hyaluronic acid (HA) were mixed in a 10:1 volume ratio to obtain a composite solution, namely HPHA complex. Next, the sodium metaperiodate was added to form a hydrogel. The samples were characterized using a zeta potential (ZP) analyzer, transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FTIR), and water contact angle (WCA). Enzyme-linked immunosorbent assay (ELISA) and toluidine blue O (TBO) staining were employed to assess the immobilization and release of VEGF and heparin. Rhodamine staining was adopted to evaluate the adhesion of platelets, fibrinogen, macrophages, and umbilical artery smooth muscle cells (UASMCs). Furthermore, CCK-8 assay was performed to evaluate the adhesion and proliferation of endothelial cells (ECs). The results revealed that compared to 0 mg/L, the particle size (PS), polydispersity index (PDI), and θ angle decreased, and the absolute value of ZP increased at 100, 200, 300, and 400 mg/L. The cumulative release (CR) of VEGF at 200 mg/L was higher to that at 0 mg/L, while that of heparin was lower (P <0.05). The platelet surface coverage rate (PSCR), relative platelet activation rate (RPAR), and fibrinogen coverage rate (FCR) at 10 and 20 days were lower and exhibited great difference to those at 0 mg/L (P <0.05). Compared to groups II and III, group I presented a markedly higher number of macrophages and UASMCs on the first and third days of culture (P <0.05). 3 days after culture, adhesion and proliferation of ECs in groups U, I, and K increased greatly (P <0.05). In conclusion, the HPHA complex prepared from HP NPs loaded with VEGF and HA in this research exhibited good stability, uniformity, hydrophilicity, and blood compatibility. It effectively blocked the adhesion of macrophages and UASMCs and promoted the proliferation of ECs, thus conforming to the mechanism of inhibiting CAR after coronary stent intervention.

Optimal mechanical interactions direct multicellular network formation on elastic substrates

Cells self-organize into functional, ordered structures during tissue morphogenesis, a process that is evocative of colloidal self-assembly into engineered soft materials. Understanding how intercellular mechanical interactions may drive the formation of ordered and functional multicellular structures is important in developmental biology and tissue engineering. Here, by combining an agent-based model for contractile cells on elastic substrates with endothelial cell culture experiments, we show that substrate deformation-mediated mechanical interactions between cells can cluster and align them into branched networks. Motivated by the structure and function of vasculogenic networks, we predict how measures of network connectivity like percolation probability and fractal dimension as well as local morphological features including junctions, branches, and rings depend on cell contractility and density and on substrate elastic properties including stiffness and compressibility. We predict and confirm with experiments that cell network formation is substrate stiffness dependent, being optimal at intermediate stiffness. We also show the agreement between experimental data and predicted cell cluster types by mapping a combined phase diagram in cell density substrate stiffness. Overall, we show that long-range, mechanical interactions provide an optimal and general strategy for multicellular self-organization, leading to more robust and efficient realizations of space-spanning networks than through just local intercellular interactions.

Nerve pathology of microangiopathy and thromboinflammation in hereditary transthyretin amyloidosis.

Despite amyloid deposition as a hallmark of hereditary transthyretin amyloidosis (ATTRv) with polyneuropathy, this pathology could not completely account for nerve degeneration. ATTRv patients frequently have vasomotor symptoms, but microangiopathy hypothesis in ATTRv was not systemically clarified. This study examined the vascular pathology of sural nerves in ATTRv patients with transthyretin (TTR) mutation of p.Ala117Ser (TTR-A97S), focusing on morphometry and patterns of molecular expression in relation to nerve degeneration. We further applied human microvascular endothelial cell (HMEC-1) culture to examine the direct effect of TTR-A97S protein on endothelial cells. In ATTRv nerves, there was characteristic microangiopathy compared to controls: increased vessel wall thickness and decreased luminal area; both were correlated with the reduction of myelinated fiber density. Among the components of vascular wall, the area of collagen IV in ATTRv nerves was larger than that of controls. This finding was validated in a cell model of HMEC-1 culture in which the expression of collagen IV was upregulated after exposure to TTR-A97S. Apoptosis contributed to the endothelial cell degeneration of microvasculatures in ATTRv endoneurium. ATTRv showed prothrombotic status with intravascular fibrin deposition, which was correlated with (1) increased tissue factor and coagulation factor XIIIA and (2) reduced tissue plasminogen activator. This cascade led to intravascular thrombin deposition, which was colocalized with upregulated p-selectin and thrombomodulin, accompanied by complement deposition and macrophages infiltration, indicating thromboinflammation in ATTRv. Microangiopathy with thromboinflammation is characteristic of advanced-stage ATTRv nerves, which provides an add-on mechanism and therapeutic target for nerve degeneration.

CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood-retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR.

Cell Cultures as a Versatile Tool in the Research and Treatment of Autoimmune Connective Tissue Diseases.

Cell cultures are an important part of the research and treatment of autoimmune connective tissue diseases. By culturing the various cell types involved in ACTDs, researchers are able to broaden the knowledge about these diseases that, in the near future, may lead to finding cures. Fibroblast cultures and chondrocyte cultures allow scientists to study the behavior, physiology and intracellular interactions of these cells. This helps in understanding the underlying mechanisms of ACTDs, including inflammation, immune dysregulation and tissue damage. Through the analysis of gene expression patterns, surface proteins and cytokine profiles in peripheral blood mononuclear cell cultures and endothelial cell cultures researchers can identify potential biomarkers that can help in diagnosing, monitoring disease activity and predicting patient's response to treatment. Moreover, cell culturing of mesenchymal stem cells and skin modelling in ACTD research and treatment help to evaluate the effects of potential drugs or therapeutics on specific cell types relevant to the disease. Culturing cells in 3D allows us to assess safety, efficacy and the mechanisms of action, thereby aiding in the screening of potential drug candidates and the development of novel therapies. Nowadays, personalized medicine is increasingly mentioned as a future way of dealing with complex diseases such as ACTD. By culturing cells from individual patients and studying patient-specific cells, researchers can gain insights into the unique characteristics of the patient's disease, identify personalized treatment targets, and develop tailored therapeutic strategies for better outcomes. Cell culturing can help in the evaluation of the effects of these therapies on patient-specific cell populations, as well as in predicting overall treatment response. By analyzing changes in response or behavior of patient-derived cells to a treatment, researchers can assess the response effectiveness to specific therapies, thus enabling more informed treatment decisions. This literature review was created as a form of guidance for researchers and clinicians, and it was written with the use of the NCBI database.