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Endothelial cells (ECs) not only form passive blood conduits but actively contribute to nutrient transport and organ homeostasis. The role of ECs in glucose homeostasis is, however, poorly understood. Here, we show that, in skeletal muscle, endothelial glucose transporter 1 (Glut1 /Slc2a1) controls glucose uptake via vascular metabolic control of muscle-resident macrophages without affecting transendothelial glucose transport. Lowering endothelial Glut1 via genetic depletion (Glut1 ΔEC) or upon a short-term high-fat diet increased angiocrine osteopontin (OPN/ Spp1) secretion. This promoted resident muscle macrophage activation and proliferation, which impaired muscle insulin sensitivity. Consequently, co-deleting Spp1 from ECs prevented macrophage accumulation and improved insulin sensitivity in Glut1 ΔEC mice. Mechanistically, Glut1- dependent endothelial glucose metabolic rewiring increased OPN in a serine metabolism-dependent fashion. Our data illustrate how the glycolytic endothelium creates a microenvironment that controls resident muscle macrophage phenotype and function and directly links resident muscle macrophages to the maintenance of muscle glucose homeostasis. [Display omitted] • EC-specific loss of Glut1 impairs insulin sensitivity in skeletal muscle • Angiocrine OPN controls resident macrophage phenotype and function • Resident macrophage accumulation and activation impair muscle insulin sensitivity • Endothelial glucose metabolic control of OPN is serine dependent Zhang et al. found that endothelial GLUT1 regulates muscle insulin sensitivity independently of transendothelial glucose transport. Lowering endothelial GLUT1 through genetic deletion or upon a short-term high-fat diet rewires endothelial glucose metabolism, thereby increasing angiocrine OPN secretion. This promotes resident macrophage accumulation and activation, which leads to muscle insulin resistance. [ABSTRACT FROM AUTHOR]

The first trimester placenta is very rarely investigated for placental vascular formation in developmental or diseased contexts. Defects in placental formation can cause heart defects in the fetus, and vice versa. Determining the causality is therefore difficult as both organs develop concurrently and express many of the same genes. Here, we performed a systematic review to determine feto-placental and coronary endothelial genes implicated in miscarriages, stillbirth and congenital heart defects (CHD) from human genome wide screening studies. 4 single cell RNAseq datasets from human first/early second trimester cardiac and placental samples were queried to generate a list of 1187 endothelial genes. This broad list was cross-referenced with genes implicated in the pregnancy disorders above. 39 papers reported feto-placental and cardiac coronary endothelial genes, totalling 612 variants. Vascular gene variants were attributed to the incidence of miscarriage (8 %), CHD (4 %) and stillbirth (3 %). The most common genes for CHD (NOTCH, DST, FBN1, JAG1, CHD4), miscarriage (COL1A1, HERC1), and stillbirth (AKAP9, MYLK), were involved in blood vessel and cardiac valve formation, with roles in endothelial differentiation, angiogenesis, extracellular matrix signaling, growth factor binding and cell adhesion. NOTCH1, AKAP12, CHD4, LAMC1 and SOS1 showed greater relative risk ratios with CHD. Many of the vascular genes identified were expressed highly in both placental and heart EC populations. Both feto-placental and cardiac vascular genes are likely to result in poor endothelial cell development and function during human pregnancy that leads to higher risk of miscarriage, congenital heart disease and stillbirth. • Placental and cardiac vascular endothelial genes are associated with pregnancy-associated conditions. • 39 studies reported significant vascular genes for congenital heart defects, miscarriage and stillbirth. • Many genes were expressed in both first trimester and early second trimester coronary and placental endothelial cells. • This data verifies known vascular genes with important roles in endothelial specification and function. • This provides new gene candidates to explore in the miscarriage, congenital heart disease and stillbirth fields. [ABSTRACT FROM AUTHOR]

Skin tissue engineering is undergoing tremendous expansion as a result from clinical needs, mandatory replacement of animal models and development of new technologies. Many approaches have been used to produce vascularized skin substitutes for grafting purposes showing the presence of capillary-like structures but with limited analysis of their in vitro maturation and plasticity. Such knowledge is however important for the development of tissue substitutes with improved implantation success as well as for validation of vascularization in vitro models, including as a readout in pharmacological analyses. For optimal interactions of cells with microenvironment and vasculature, we here used a cell sheet approach consisting in the sole production of matrix by the cells. In this context, we limited the density of endothelial cells seeded for self-assembly and rather relied on the stimulation of angiogenesis for the development of an extensive connected microvascular-like network. After detailed characterization of this network, we challenged its plasticity both during and after establishment of the skin substitute. We show that fine tuning of VEGF concentration and time of application differentially affects formation of capillary-like structures and their perivascular coverage. Furthermore, we performed a deep wound assay that displayed tissue repair and angiogenesis with unique characteristics of the physiological process. These studies demonstrate the importance of cell-derived microenvironment for the establishment of mature yet dynamic vascularized skin models allowing a wide range of pharmacological and basic investigations. The significant advancements in organ-on-chips and tissue engineering call for more relevant models including microvascularization with remodeling potential. While vascularized skin substitutes have been developed for years, focus has primarily been on the impact of microvascularization on implantation rather than on its in vitro characterization. We here developed a cell sheet-based vascularized skin substitute relying on angiogenesis, i.e. growth of vessel-like structures within the 3D model, rather than solely on endothelial cell self-assembly. We then characterized :1/ vascularization after modulation of angiogenic factor VEGF during the substitute construction; -2/ angiogenesis associated to tissue repair after deep mechanical wounding. These studies establish a solid physiologically relevant model for further investigation of skin cell interactions and in vitro wound healing. [Display omitted] [ABSTRACT FROM AUTHOR]

BACKGROUND: Drp1 (dynamin-related protein 1), a large GTPase, mediates the increased mitochondrial fission, which contributes to hyperproliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension (PAH). We developed a potent Drp1 GTPase inhibitor, Drpitor1a, but its specificity, pharmacokinetics, and efficacy in PAH are unknown. METHODS: Drpitor1a's ability to inhibit recombinant and endogenous Drp1 GTPase was assessed. Drpitor1a's effects on fission were studied in control and PAH human pulmonary artery smooth muscle cells (hPASMC) and blood outgrowth endothelial cells (BOEC). Cell proliferation and apoptosis were studied in hPASMC. Pharmacokinetics and tissue concentrations were measured following intravenous and oral drug administration. Drpitor1a's efficacy in regressing monocrotaline-PAH was assessed in rats. In a pilot study, Drpitor1a reduced PA remodeling only in females. Subsequently, we compared Drpitor1a to vehicles in control and monocrotaline-PAH females. RESULTS: Drp1 GTPase activity was increased in PAH hPASMC. Drpitor1a inhibited the GTPase activity of recombinant and endogenous Drp1 and reversed the increased fission, seen in PAH hPASMC and PAH BOEC. Drpitor1a inhibited proliferation and induced apoptosis in PAH hPASMC without affecting electron transport chain activity, respiration, fission/ fusion mediator expression, or mitochondrial Drp1 translocation. Drpitor1a did not inhibit proliferation or alter mitochondrial dynamics in normal hPASMC. Drpitor1a regressed monocrotaline-PAH without systemic vascular effects or toxicity. CONCLUSIONS: Drpitor1a is a specific Drp1 GTPase inhibitor that reduces mitochondrial fission in PAH hPASMC and PAH BOEC. Drpitor1a reduces proliferation and induces apoptosis in PAH hPASMC and regresses monocrotaline-PAH. Drp1 is a therapeutic target in PAH, and Drpitor1a is a potential therapy with an interesting therapeutic sexual dimorphism. [ABSTRACT FROM AUTHOR]

BACKGROUND: Folate intake during pregnancy is essential for fetal development and maternal health. However, the specific effects of folic acid (FA) and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) on the prevention and treatment of hypertensive disorders of pregnancy remain unclear. We investigated whether FA and 5-MTHF have different effects on endothelial cell tetrahydrobiopterin (BH4) metabolism in pregnancy and the possible consequences for endothelial NO generation, maternal blood pressure, and fetal growth. METHODS: We analyzed the maternal blood pressure in pregnant wild-type (Gch1fl/fl) and Gch1fl/fl Tie2cre mice treated with either FA or 5-MTHF starting before pregnancy, mid-pregnancy or late pregnancy. BH4, superoxide, and NO bioavailability were determined in mouse and human models of endothelial cell BH4 deficiency by high-performance liquid chromatography. RESULTS: In vitro studies in mouse and human endothelial cells showed that treatment with 5-MTHF, but not FA, elevated BH4 levels, reduced superoxide production, and increased NO synthase activity. In primary endothelial cells isolated from women with hypertensive pregnancies, exposure to 5-MTHF, but not FA, restored the reduction in BH4 levels and NO synthase activity. In vivo studies in mice revealed that oral treatment with 5-MTHF, but not FA, prevented and treated hypertension in pregnancy when administered either before or during pregnancy, respectively, and normalized placental and fetal growth restriction if administered from mid-gestation onward. CONCLUSIONS: Collectively, these studies identify a critical role for 5-MTHF in endothelial cell function in pregnancy, related to endothelial cell BH4 availability and NO synthase activity. Thus, 5-MTHF represents a novel therapeutic agent that may potentially improve endothelial function in hypertensive disorders of pregnancy by targeting endothelial cell BH4. [ABSTRACT FROM AUTHOR]

Excessive subendothelial retention of oxidized low-density lipoprotein (oxLDL) and subsequent oxLDL engulfment by macrophages leads to the formation of foam cells and the development of atherosclerosis. Our previous study showed that the plasma level of sialic acid-binding immunoglobulin-like lectin 5 (Siglec-5) was a novel biomarker for the prognosis of atherosclerosis in diabetic patients. However, the role and underlying mechanisms of Siglec-5 in atherosclerosis have not been elucidated. The interaction between oxLDL and Siglec-5 was detected by fluorescence colocalization and coimmunoprecipitation. The effect of oxLDL on Siglec-5 expression was detected in endothelial cells and macrophages, and the effect of Siglec-5 on oxLDL transcytosis and uptake was investigated. Siglec-5 was overexpressed in mice using recombinant adeno-associated virus vector serotype 9 (rAAV9-Siglec-5) to evaluate the effect of Siglec-5 on oxLDL uptake and atherogenesis in vivo. In addition, the effects of Siglec-5 antibodies and soluble Siglec-5 proteins on oxLDL transcytosis and uptake and their role in atherogenesis were investigated in vivo and in vitro. We found that oxLDL interacted with Siglec-5 and that oxLDL stimulated the expression of Siglec-5. Siglec-5 promotes the transcytosis and uptake of oxLDL, while both anti-Siglec-5 antibodies and soluble Siglec-5 protein attenuated oxLDL transcytosis and uptake. Interestingly, overexpression of Siglec-5 by recombinant adeno-associated viral vector serotype 9 (rAAV9-Siglec-5) promoted the retention of oxLDL in the aorta of C57BL/6 mice. Moreover, overexpression of Siglec-5 significantly accelerated the formation of atherosclerotic lesions in Apoe−/− mice. Moreover, both anti-Siglec-5 antibodies and soluble Siglec-5 protein significantly alleviated the retention of oxLDL in the aorta of rAAV9-Siglec-5-transfected C57BL/6 mice and the formation of atherosclerotic plaques in rAAV9-Siglec-5-transfected Apoe−/− mice. Our results suggested that Siglec-5 was a novel receptor that mediated oxLDL transcytosis and promoted the formation of foam cells. Interventions that inhibit the interaction between oxLDL and Siglec-5, including anti-Siglec-5 antibody or soluble Siglec-5 protein treatment, may provide novel therapeutic strategies in treating atherosclerosis. [ABSTRACT FROM AUTHOR]

The endothelial-mesenchymal transition (EndMT) of endothelial progenitor cells (EPCs) plays a notable role in pathological vascular remodeling. Emerging evidence indicated that long noncoding RNA-regulator of reprogramming (linc-ROR) can promote epithelial-mesenchymal transition (EMT) in a variety of cancer cells. Nevertheless, the function of linc-ROR in EPC EndMT has not been well elucidated. The present study investigated the effect and possible mechanisms of function of linc-ROR on the EndMT of EPCs. A linc-ROR overexpression lentiviral vector (LV-linc-ROR) or a linc-ROR short hairpin RNA lentiviral vector (LV-shlinc-ROR) was used to up or downregulate linc-ROR expression in EPCs isolated from human umbilical cord blood. Functional experiments demonstrated that LV-linc-ROR promoted the proliferation and migration of EPCs, but inhibited EPC angiogenesis in vitro. In the meantime, reverse transcriptionquantitative PCR and western blotting results showed that the expression of the endothelial cell markers vascular endothelialcadherin and CD31 was decreased, while the expression of the mesenchymal cell markers α-smooth muscle actin and SM22α was increased at both mRNA and protein levels in LV-linc-RORtreated EPCs, indicating that linc-ROR induced EPC EndMT. Mechanistically, the dual-luciferase reporter assay demonstrated that microRNA (miR/miRNA)-145 was a direct target of linc-ROR, and miR-145 binds to the 3'-untranslated region of Smad3. Moreover, LV-shlinc-ROR increased the expression of miR-145, but decreased the expression of Smad3. In conclusion, linc-ROR promotes EPC EndMT, which may be associated with the miR- 145/Smad3 signaling pathway. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis poses a significant health threat with very limited therapeutic options available. In this study, we reported the enhanced expression of mesenchymal homobox 1 (MEOX1) in pulmonary fibrosis patients, especially in their fibroblasts and endothelial cells, and confirmed MEOX1 as a central orchestrator in the activation of profibrotic genes. By high-throughput screening, we identified Ailanthone (AIL) from a natural compound library as the first small molecule capable of directly targeting and suppressing MEOX1. AIL demonstrated the ability to inhibit both the activation of fibroblasts and endothelial-to-mesenchymal transition of endothelial cells when challenged by transforming growth factor- β 1 (TGF- β 1). In an animal model of bleomycin-induced pulmonary fibrosis, AIL effectively mitigated the fibrotic process and restored respiratory functions. Mechanistically, AIL acted as a suppressor of MEOX1 by disrupting the interaction between the transcription factor JUN and the promoter of MEOX1, thereby inhibiting MEOX1 expression and activity. In summary, our findings pinpointed MEOX1 as a cell-specific and clinically translatable target in fibrosis. Moreover, we demonstrated the potent anti-fibrotic effect of AIL in pulmonary fibrosis, specifically through the suppression of JUN-dependent MEOX1 activation. MEOX1 is a central orchestrator during pulmonary fibrosis and promotes fibroblasts activation and EndMT. High-throughput screening identifies Ailanthone as the first direct suppressor of MEOX1 by disrupting the interaction between JUN and MEOX1 promoter. [Display omitted] [ABSTRACT FROM AUTHOR]

BACKGROUND: Hypoxia-induced pulmonary hypertension (HPH) is a T helper 17 cell response-driven disease, and PD-1 (programmed cell death 1)/PD-L1 (programmed cell death-ligand 1) inhibitor-associated pulmonary hypertension has been reported recently. This study is designed to explore whether the PD-1/PD-L1 pathway participates in HPH via regulating endothelial dysfunction and T helper 17 cell response. METHODS: Lung tissue samples were obtained from eligible patients. Western blotting, immunohistochemistry, and immunofluorescence techniques were used to assess protein expression, while immunoprecipitation was utilized to detect ubiquitination. HPH models were established in C57BL/6 WT (wild-type) and PD-1-/-mice, followed by treatment with PD-L1 recombinant protein. Adeno-associated virus vector delivery was used to upregulate PD-L1 in the endothelial cells. Endothelial cell function was assessed through assays for cell angiogenesis and adhesion. RESULTS: Expression of the PD-1/PD-L1 pathway was downregulated in patients with HPH and mouse models, with a notable decrease in PD-L1 expression in endothelial cells compared with the normoxia group. In comparison to WT mice, PD-1-/-mice exhibited a more severe HPH phenotype following exposure to hypoxia, However, administration of PDL1 recombinant protein and overexpression of PD-L1 in lung endothelial cells mitigated HPH. In vitro, blockade of PDL1 with a neutralizing antibody promoted endothelial cell angiogenesis, adhesion, and pyroptosis. Mechanistically, hypoxia downregulated PD-L1 protein expression through ubiquitination. Additionally, both in vivo and in vitro, PD-L1 inhibited T helper 17 cell response through the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in HPH. CONCLUSIONS: PD-1/PD-L1 plays a role in ameliorating HPH development by inhibiting T helper 17 cell response through the PI3K/AKT/mTOR pathway and improving endothelial dysfunction, suggesting a novel therapeutic indication for PD-1/PD-L1-based immunomodulatory therapies in the treatment of HPH. [ABSTRACT FROM AUTHOR]

The development of high-throughput anticancer drug screening methods using patient-derived cancer cell (PDC) lines that maintain their original characteristics in an in vitro three-dimensional (3D) culture system poses a significant challenge to achieving personalized cancer medicine. Because stromal tissue plays a critical role in the composition and maintenance of the cancer microenvironment, in vitro 3D-culture using reconstructed stromal tissues has attracted considerable attention. Here, a simple and unique in vitro 3D-culture method using heparin and collagen together with fibroblasts and endothelial cells to fabricate vascularized 3D-stromal tissues for in vitro culture of PDCs is reported. Whereas co-treatment with bevacizumab, a monoclonal antibody against vascular endothelial growth factor, and 5-fluorouracil significantly reduced the survival rate of 3D-cultured PDCs to 30%, separate addition of each drug did not induce comparable strong cytotoxicity, suggesting the possibility of evaluating the combined effect of anticancer drugs and angiogenesis inhibitors. Surprisingly, drug evaluation using eight PDC lines with the 3D-culture method resulted in a drug efficacy concordance rate of 75% with clinical outcomes. The model is expected to be applicable to in vitro throughput drug screening for the development of personalized cancer medicine. To replicate the cancer microenvironment, we constructed a cancer-stromal tissue model in which cancer cells are placed above and inside stromal tissue with vascular network structures derived from vascular endothelial cells in fibroblast tissue using CAViTs method. Using this method, we were able to reproduce the invasion and metastasis processes of cancer cells observed in vivo. Using patient-derived cancer cells, we assessed the possibility of evaluating the combined effect with an angiogenesis inhibitor. Further, primary cancer cells also grew on the stromal tissues with the normal medium. These data suggest that the model may be useful for new in vitro drug screening and personalized cancer medicine. [Display omitted] [ABSTRACT FROM AUTHOR]

This study aimed to establish a blood vessel model within a hollow fiber bioreactor to evaluate the impact of high and fluctuating glucose levels on human umbilical vein endothelial cells (HUVECs) under laminar shear stress (LSS). HUVECs were cultured for 48 h in normal (5 mM), high (20 mM), and variable (20 mM / 5 mM alternating every 24 h) glucose concentrations under LSS of 0.66 Pa. An automated medium replacement system was developed. The control cultures remained static. The analysis included cell viability via cytometric analysis, glucose consumption, lactate production via electroenzymatic methods, and the expression of 21 genes via qPCR. The percentage of apoptotic cells did not significantly differ across glucose concentrations under LSS. HUVECs favor glycolysis for energy regardless of LSS. Under LSS, the IL1B , CCL2 , and SELE genes were upregulated under high-glucose conditions and downregulated under variable-glucose conditions. A few other genes related to inflammation, oxidative stress, cell adhesion and apoptosis were upregulated under high-glucose conditions. In conclusion, using the blood vessel model we effectively examined the impact of glucose profiles on HUVECs under LSS in a device replicating the cylindrical geometry of blood vessels. LSS and tubular cell arrangement might mitigate the adverse effects of variable glucose on endothelial cells. [ABSTRACT FROM AUTHOR]

Exosomes or small extracellular vesicles (sEVs) are present in the blood of pregnant mice and considered to be involved in pregnancy physiology. Although sEVs in pregnant periods are proposed to be derived from placentas, sEVs-producing cells are not well known in mouse placentas. We studied the dynamics and localization of sEVs in pregnant serum and placentas, and examined gestational variation of microRNA (miRNA). Serums and placentas were collected from non-pregnant (NP) and pregnant mice throughout the entire gestational day (Gd). EVs were purified from serums and total RNA was isolated from EVs. Nanoparticle-tracking assay (NTA) revealed that the rates of sEVs in EVs are 53% at NP, and increased to 80.1% at Gd 14.5 and 97.5% at Gd 18.5. Western blotting on EVs showed positive reactivity to the tetraspanin markers and clarified that the results using anti-CD63 antibody were most consistent with the sEVs appearance detected by NTA. Serum EVs also showed a positive reaction to the syncytiotrophoblast marker, syncytin-1. Immunohistostaining using anti-CD63 antibody showed positive reactions in mouse placentas at the syncytiotrophoblasts and endothelial cells of the fetal capillaries. Quantitative PCR revealed that significantly higher amounts of miRNAs were included in the sEVs of Gd 18.5. Our results suggested that sEVs are produced in the mouse placenta and transferred to maternal or fetal bloodstreams. sEVs are expected to have a miRNA-mediated physiological effect and become useful biomarkers reflecting the pregnancy status. [ABSTRACT FROM AUTHOR]

Cytokines play a crucial role in the structure and function of blood vessels in hypertension. Hypertension damages blood vessels by mechanisms linked to shear forces, activation of the renin-angiotensin-aldosterone and sympathetic nervous systems, oxidative stress, and a proinflammatory milieu that lead to the generation of neoantigens and damage-associated molecular patterns, ultimately triggering the release of numerous cytokines. Damage-associated molecular patterns are recognized by PRRs (pattern recognition receptors) and activate inflammatory mechanisms in endothelial cells, smooth muscle cells, perivascular nerves, and perivascular adipose tissue. Activated vascular cells also release cytokines and express factors that attract macrophages, dendritic cells, and lymphocytes to the blood vessels. Activated and differentiated T cells into Th1, Th17, and Th22 in secondary lymphoid organs migrate to the vessels, releasing specific cytokines that further contribute to vascular dysfunction and remodeling. This chronic inflammation alters the profile of endothelial and smooth muscle cells, making them dysfunctional. Here, we provide an overview of how cytokines contribute to hypertension by impacting the vasculature. Furthermore, we explore clinical perspectives about the modulation of cytokines as a potential therapeutic intervention to specifically target hypertension-linked vascular dysfunction. [ABSTRACT FROM AUTHOR]

Statment of the Problem: Podoplanin can indicate the lymphangiogenesis. On the other hand, lymphangiogenesis affects the biological behavior of lesions. The clinical behavior of odontogenic keratocysts (OKC) and dentigerous cysts (DC) is different. Purpose: This study aimed to evaluate the immunohistochemical expression of podoplanin and to investigate lymphangiogenesis in OKCs as compared to DCs. Materials and Method: In this experimental laboratory study, sixty paraffined blocks, including 30 OKC and 30 DC samples, were examined in this study, all of which were histopathologically non-inflamed. To evaluate lymphangiogenesis, the immunohistochemical reaction of D2-40 was evaluated via cytoplasmic and membrane staining of lymphatic endothelial cells. The expression of podoplanin in the epithelial cells of two cyst groups was also examined. To analyze the collected data and compare the results between the two groups of cysts, independent samples t-test, Mann-Whitney U test, and Chi-square test were performed in SPSS version 22. The significance level was set at 0.05. Results: The mean lymph node count and podoplanin expression were significantly higher in the OKC epithelium as compared to DC (p< 0.001). Based on the results, 90% of OKC samples and 43.3% of DC samples showed grade 3 staining. Conclusion: The rate of lymphangiogenesis and podoplanin expression in the epithelium were higher in OKCs compared to DCs. According to the results, the expression of podoplanin may be a useful marker for determining the invasiveness and proliferation of OKC. [ABSTRACT FROM AUTHOR]

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho‐associated coiled‐coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)‐inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc‐CreERT2) were generated and administered a 5‐day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double‐floxed control mice. On Day 3 post‐TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post‐TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)‐cadherin, β‐catenin, and p120‐catenin at cell–cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE‐cadherin/β‐catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell–cell adhesion. [ABSTRACT FROM AUTHOR]

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin‐1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)—particularly EVs of vascular origin—could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)‐derived small EVs (<200 nm; HU‐sEVs) were investigated as a potential pro‐angiogenic agent. Treating mesenchymal stem cells (MSCs) and mature HUVEC cells with HU‐sEVs induced their tube formation under in vitro conditions and significantly increased the expression of angiogenesis‐related genes, such as Ang1, VEGF, Flk‐1 (VEGF receptor 2), Flt‐1 (VEGF receptor 1), and vWF (von Willebrand Factor), in a dose‐dependent manner. These results indicate that HU‐sEVs take part in angiogenesis activities in physiological systems, and suggest endothelial EVs as a potential therapeutic candidate for the treatment of angiogenesis‐related diseases. [ABSTRACT FROM AUTHOR]

Antibody-mediated rejection (ABMR) remains a significant cause of allograft failure in patients with kidney transplantation. Despite successful strategies to treat ABMR, irreversible injury in the form of transplant glomerulopathy commonly manifests months to years later. Treatment for ABMR is not standardized, and treatment guidelines vary among different transplant centers. The outcomes for patients at risk for ABMR have not significantly changed over the years. This is largely because of an incomplete understanding of the mechanisms of injury responsible for different phenotypes of ABMR. Establishing relevant surrogate endpoints to facilitate more informative studies will likely allow for a more accurate determination of prognosis and pave the way for new and effective therapeutic approaches. This article aims to summarize the current understanding of the mechanism of injury in ABMR and highlight current diagnostic approaches and treatment strategies. [ABSTRACT FROM AUTHOR]

BACKGROUND: High blood pressure has been suggested to accelerate vascular injury-induced neointimal formation and progression. However, little is known about the intricate relationships between vascular injury and hypertension in the context of arterial remodeling. METHODS: Single-cell RNA-sequencing analysis was used to depict the cell atlas of carotid arteries of Wistar Kyoto rats and spontaneously hypertensive rats with or without balloon injury. RESULTS: We found that hypertension significantly aggravated balloon injury-induced arterial stenosis. A total of 36 202 cells from carotid arteries with or without balloon injury were included in single-cell RNA-sequencing analysis. Cell composition analysis showed that vascular injury and hypertension independently induced distinct aortic cell phenotypic alterations including immune cells, endothelial cells (ECs), and smooth muscle cells. Specifically, our data showed that injury and hypertension-induced specific EC phenotypic alterations, and revealed a transition from functional ECs to hypermetabolic, and eventually dysfunctional ECs in hypertensive rats upon balloon injury. Importantly, our data also showed that vascular injury and hypertension-induced different smooth muscle cell phenotypic alterations, characterized by deferential expression of synthetic signatures. Interestingly, pathway analysis showed that dysregulated metabolic pathways were a common feature in monocytes/macrophages, ECs, and smooth muscle cells in response to injury and hypertension. Functionally, we demonstrate that inhibition of mitochondrial respiration significantly ameliorated injury-induced neointimal formation in spontaneously hypertensive rats. CONCLUSIONS: This study provides the cell landscape changes of the main aortic cell phenotypic alterations in response to injury and hypertension. Our findings suggest that targeting cellular mitochondrial respiration could be a novel therapeutic for patients with hypertension undergoing vascular angioplasty. [ABSTRACT FROM AUTHOR]

The effects of helical flow in a blood vessel are investigated in a dynamic flow generator using surface acoustic wave (SAW) in the microfluidic device. The SAW, generated by an interdigital transducer (IDT), induces acoustic streaming, resulting in a stable and consistent helical flow pattern in microscale channels. This approach allows rapid development of helical flow within the channel without directly contacting the medium. The precise design of the window enables the creation of distinct unidirectional vortices, which can be controlled by adjusting the amplitude of the SAW. Within this device, optimal operational parameters of the dynamic flow generator to preserve the integrity of endothelial cells are found, and in such settings, the actin filaments within the cells are aligned to the desired state. Our findings reveal that intracellular Ca2+ concentrations vary in response to flow conditions. Specifically, comparable maximum intensity and graphical patterns were observed between low-flow rate helical flow and high-flow rate Hagen-Poiseuille flow. These suggest that the cells respond to the helical flow through mechanosensitive ion channels. Finally, adherence of monocytes is effectively reduced under helical flow conditions in an inflammatory environment, highlighting the atheroprotective role of helical flow. Helical flow in blood vessels is well known to prevent atherosclerosis. However, despite efforts to replicate helical flow in microscale channels, there is still a lack of in vitro models which can generate helical flow for analyzing its effects on the vascular system. In this study, we developed a method for generating steady and constant helical flow in microfluidic channel using acoustofluidic techniques. By utilizing this dynamic flow generator, we were able to observe the atheroprotective aspects of helical flow in vitro, including the enhancement of calcium ion flux and reduction of monocyte adhesion. This study paves the way for an in vitro model of dynamic cell culture and offers advanced investigation into helical flow in our circulatory system. [Display omitted] [ABSTRACT FROM AUTHOR]

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The incorporation of cobalt ions into the composition of bioactive glasses has emerged as a strategy of interest for bone regeneration purposes. In the present work, we have designed a set of bioactive mesoporous glasses SiO 2 -CaO-P 2 O 5 -CoO (Co-MBGs) with different amounts of cobalt. The physicochemical changes introduced by the Co2+ ion, the in vitro effects of Co-MBGs on preosteoblasts and endothelial cells and their in vivo behaviour using them as bone grafts in a sheep model were studied. The results show that Co2+ ions neither destroy mesoporous ordering nor inhibit in vitro bioactive behaviour, exerting a dual role as network former and modifier for CoO concentrations above 3 % mol. On the other hand, the activity of Co-MBGs on MC3T3-E1 preosteoblasts and HUVEC vascular endothelial cells is dependent on the concentration of CoO present in the glass. For low Co-MBGs concentrations (1mg/ml) cell viability is not affected, while the expression of osteogenic (ALP, RUNX2 and OC) and angiogenic (VEGF) genes is stimulated. For Co-MBGs concentration of 5 mg/ml, cell viability decreases as a function of the CoO content. In vivo studies show that the incorporation of Co2+ ions to the MBGs improves the bone regeneration activity of these materials, despite the deleterious effect that this ion has on bone-forming cells for any of the Co-MBG compositions studied. This contradictory effect is explained by the marked increase in angiogenesis that takes place inside the bone defect, leading to an angiogenesis-osteogenesis coupling that compensates for the partial decrease in osteoblast cells. The development of new bone grafts implies to address the need for osteogenesis-angiogenesis coupling that allows bone regeneration with viable tissue in the long term. In this sense the incorporation of cobalt ions into the composition of bioactive glasses has emerged as a strategy of great interest in this field. Due to the potential cytotoxic effect of cobalt ions, there is an important controversy regarding the suitability of their incorporation in bone grafts. In this work, we address this controversy after the implantation of cobalt-doped mesoporous bioactive glasses in a sheep model. The incorporation of cobalt ions in bioactive glasses improves the bone regeneration ability of these bone grafts, due to enhancement of the angiogenesis-osteogenesis coupling. [Display omitted] [ABSTRACT FROM AUTHOR]

Studies have highlighted the significant involvement of kidney pericytes in renal fibrosis. Kidney pericytes, classified as interstitial mesenchymal cells, are extensively branched, collagen-producing cells that closely interact with endothelial cells. This article aims to provide an overview of the recent advancements in understanding the physiological functions of pericytes and their roles in kidney diseases. In a healthy kidney, pericytes have essential physiological function in angiogenesis, erythropoietin (EPO) production, and the regulation of renal blood flow. Nevertheless, pericyte–myofibroblast transition has been identified as the primary cause of disease progression in acute kidney injury (AKI)-to-chronic kidney disease (CKD) continuum. Our recent research has demonstrated that hypoxia-inducible factor-2α (HIF-2α) regulates erythropoietin production in pericytes. However, this production is repressed by EPO gene hypermethylation and HIF-2α downregulation which were induced by transforming growth factor-β1-activated DNA methyltransferase and activin receptor-like kinase-5 signaling pathway during renal fibrosis, respectively. Additionally, AKI induces epigenetic modifications in pericytes, rendering them more prone to extracellular matrix production, cell migration and proliferation, thereby contributing to subsequent capillary rarefaction and renal fibrosis. Further investigation into the specific functions and roles of different subpopulations of pericytes may contribute for the development of targeted therapies aimed at attenuating kidney disease and mitigating their adverse effects. [ABSTRACT FROM AUTHOR]

Stress and illness connection is complex and involves multiple physiological systems. Panax ginsengs, reputed for their broad-spectrum "cure-all" effect, are widely prescribed to treat stress and related illnesses. However, the identity of ginseng's "cure-all" medicinal compounds that relieve stress remains unresolved. Here, we identify ginsentides as the principal bioactives that coordinate multiple systems to restore homeostasis in response to stress. Ginsentides are disulfide-rich, cell-penetrating and proteolytic-stable microproteins. Using affinity-enrichment mass spectrometry target identification together with in vitro , ex vivo and in vivo validations, we show that highly purified or synthetic ginsentides promote vasorelaxation by producing nitric oxide through endothelial cells via intracellular PI3K/Akt signaling pathway, alleviate α 1-adrenergic receptor overactivity by reversing phenylephrine-induced constriction of aorta, decrease monocyte adhesion to endothelial cells via CD166/ESAM/CD40 and inhibit P2Y12 receptors to reduce platelet aggregation. Orally administered ginsentides were effective in animal models to reduce ADP-induced platelet aggregation, to prevent collagen and adrenaline-induced pulmonary thrombosis as well as anti-stress behavior of tail suspension and forced swimming tests in mice. Together, these results strongly suggest that ginsentides are the principal panacea compounds of ginsengs because of their ability to target multiple extra- and intra-cellular proteins to reverse stress-induced damages. This study shows ginsentides are "first-in-class" bioactive peptides from ginseng with a variety of functions that answers part of the "cure-all" functions of ginseng, particularly for cardiovascular health and stress. [Display omitted] [ABSTRACT FROM AUTHOR]

The ex vivo endothelialization of small diameter vascular prostheses can prolong their patency. Here, we demonstrate that heterotypic interactions between human adipose tissue-derived endothelial cells and perivascular cells can be exploited to accelerate the endothelialization of an electrospun ionomeric polyurethane scaffold. The scaffold was used to physically separate endothelial cells from perivascular cells to prevent their diffuse neo-intimal hyperplasia and spontaneous tubulogenesis, yet enable their paracrine cross-talk to accelerate the integration of the endothelial cells into a temporally stable endothelial lining of a continuous, elongated, and aligned morphology. Perivascular cells stimulated endothelial basement membrane protein production and suppressed their angiogenic and inflammatory activation to accelerate this biomimetic morphogenesis of the endothelium. These findings demonstrate the feasibility and underscore the value of exploiting heterotypic interactions between endothelial cells and perivascular cells for the fabrication of an endothelial lining intended for small diameter arterial reconstruction. Adipose tissue is an abundant, accessible, and uniquely dispensable source of endothelial cells and perivascular cells for vascular tissue engineering. While their spontaneous self-assembly into microvascular networks is routinely exploited for the vascularization of engineered tissues, it threatens the temporal stability of an endothelial lining intended for small diameter arterial reconstruction. Here, we demonstrate that an electrospun polyurethane scaffold can be used to physically separate endothelial cells from perivascular cells to prevent their spontaneous capillary morphogenesis, yet enable their cross-talk to promote the formation of a stable endothelium. Our findings demonstrate the feasibility of engineering an endothelial lining from human adipose tissue, poising it for the rapid ex vivo endothelialization of small diameter vascular prostheses in an autologous, patient-specific manner. [Display omitted] [ABSTRACT FROM AUTHOR]

Nano-induced endothelial leakiness (NanoEL) can improve the ability of nanoparticles (NPs) to enter the tumor environment, nevertheless, it can inadvertently trigger adverse effects such as tumor metastasis. To overcome these concerns, it becomes important to develop a NPs design strategy that capitalizes on the NanoEL effect while averting unwanted side effects during the drug delivery process. Herein, we introduce the PLGA-ICG-PEI-Ang1@M NP which has a core comprising poly (lactic-co-glycolic acid) (PLGA) and the inner shell with a highly positively charged polyethyleneimine (PEI) and the anti-permeability growth factor Angiopoietin 1 (Ang1), while the outer shell is camouflaged with a Jurkat cell membrane. During the drug delivery process, our NPs exhibit their capability to selectively target and penetrate endothelial cell layers. Once the NPs penetrate the endothelial layer, the proton sponge effect triggered by PEI in the acidic environment surrounding the tumor site can rupture the cell membrane on the NPs' surface. This rupture, in turn, enables the positively charged Ang1 to be released due to the electrostatic repulsion from PEI and the disrupted endothelial layer can be restored. Consequently, the designed NPs can penetrate endothelial layers, promote the cell layer recovery, restrict the tumor metastasis, and facilitate efficient cancer therapy. • This work develops a NP design strategy that capitalizes on the nano-induced endothelial leakiness (NanoEL) effect while averting unwanted side effects such as tumor metastasis during the drug delivery process. • The rational design of core-double shell PLGA-ICG-PEI-Ang1@M NP enables enhanced drug transport across the endothelial barrier inherited from both membrane-derived tumor homing and NanoEL effect brought by NPs. • The controlled release of angiopoietin-1 in response to the tumor microenvironment allows a timely recovery of endothelial cell leakiness which can avert unwanted side effects due to the uncontrolled and persistent leakiness. • The designed NPs can penetrate endothelial layers, expedite cell layer recovery, curtail tumor metastasis, and facilitate efficient cancer therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Essential hypertension, a multifaceted disorder, is a worldwide health problem. A complex network of genetic, epigenetic, physiological, and environmental components regulates blood pressure (BP), and any dysregulation of this network may result in hypertension. Growing evidence suggests a role for epigenetic factors in BP regulation. Any alterations in the expression or functions of these epigenetic regulators may dysregulate various determinants of BP, thereby promoting the development of hypertension. Histone posttranslational modifications are critical epigenetic regulators that have been implicated in hypertension. Several studies have demonstrated a clear association between the increased expression of some histone-modifying enzymes, especially HDACs (histone deacetylases), and hypertension. In addition, treatment with HDAC inhibitors lowers BP in hypertensive animal models, providing an excellent opportunity to design new drugs to treat hypertension. In this review, we discuss the potential contribution of different histone modifications to the regulation of BP. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV), important messengers in intercellular communication, can load and transport various bioactive components and participate in different biological processes. We previously isolated glioma human endothelial cells (GhECs) and found that GhECs, rather than normal human brain endothelial cells (NhECs), exhibit specific enrichment of MYO1C into EVs and promote the migration of glioma cells. In this study, we explored the mechanism by which MYO1C is secreted into EVs. We report that such secretion is dependent on RAB31, RAB27B, and FAS. When expression of RAB31 increases, MYO1C is enriched in secretory EVs. Finally, we identified an EV export mechanism for MYO1C that promotes glioma cell invasion and is dependent on RAB31 in GhECs. In summary, our data indicate that the knockdown of RAB31 can reduce enrichment of MYO1C in extracellular vesicles, thereby attenuating the promotion of glioma cell invasion by GhEC‐EVs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Endothelial dysfunction plays a crucial role in aortic remodeling. Aerobic glycolysis and endothelial-to-mesenchymal transition (EndoMT) have, respectively, been suggested to contribute to endothelial dysfunction in many cardiovascular diseases. Here, we tested the hypothesis that glycolytic reprogramming is critical for EndoMT induction in aortic remodeling through an epigenetic mechanism mediated by a transcriptional corepressor CtBP1 (C-terminal binding protein 1), a sensor of glycolysis-derived NADH. METHODS: EndoMT program, aortic remodeling, and endothelial expression of the glycolytic activator PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) were evaluated in Ang (angiotensin) II-infused mice. Mice with endothelial-specific Pfkfb3 deficiency or CtBP1 inactivation, immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assay were employed to elucidate whether and how PFKFB3/CtBP1 epigenetically controls EndoMT. RESULTS: The EndoMT program and increased endothelial PFKFB3 expression were induced in remodeled thoracic aortas. In TGF-ß (transforming growth factor-ß)-treated human endothelial cells, activated SMAD2/3 (SMAD Family Member 2/3) transcriptionally upregulated PFKFB3 expression. In turn, the TGF-ß/SMAD signaling and EndoMT were compromised by silencing or inhibition of PFKFB3. Mechanistic studies revealed that PFKFB3-mediated glycolysis increased NADH content and activated the NADH-sensitive CtBP1. Through interaction with the transcription repressor E2F4 (E2F Transcription Factor 4), CtBP1 enhanced E2F4-mediated transcriptional repression of SMURF2 (SMAD ubiquitin regulatory factor 2), a negative regulator of TGF-ß/SMAD2 signaling. Additionally, EC-specific Pfkfb3 deficiency or CtBP1 inactivation in mice led to attenuated Ang II-induced aortic remodeling. CONCLUSIONS: Our results demonstrate a glycolysis-mediated positive feedback loop of the TGF-ß signaling to induce EndoMT and indicate that therapeutically targeting endothelial PFKFB3 or CtBP1 activity could provide a basis for treating EndoMT-linked aortic remodeling. [ABSTRACT FROM AUTHOR]

The human cornea is composed of five layers, with the stroma layer being the thickest and the endothelium layer being the densest. The endothelium layer contains hexagonally structured cells, and disturbances in these cells occur in various dystrophies, including Fuch's dystrophy (FD), advanced Fuch's dystrophy (AFD), posterior polymorphous corneal dystrophy, Irido corneal dystrophy (ICD), mild polymegathism, and corneal guttata (CG). Thirteen (13) images were obtained from a specular microscope, capturing different dystrophies in various patients. These images were processed using the Artificial Intelligent Convolution Filter (AICF) algorithm. The algorithm extracted several parameters from the endothelium layer, including mean cell area, elongation of endothelial cells, Heywood circularity, compactness, hexagonality, standard deviation, and coefficient of variation. The mean cell area of images I1 to I13 ranged from 79.5 µm² to 3485 µm². The elongation of endothelial cells varied between 3.11 µm² and 3.98 µm². The compactness factor of endothelial cells ranged from 0.62 µm² to 0.93 µm², while Heywood circularity factor ranged from 0.8 µm² to 1.98 µm². The coefficient of variation spanned from 10 µm² to 99 µm², and the hexagonality of endothelial cells ranged from 46.6 µm² to 65.2 µm². These statistical parameters provide a meticulous representation of the healthy condition of the endothelial layer. [ABSTRACT FROM AUTHOR]

Lung inflammation is an essential inducer of various diseases and is closely related to pulmonary-endothelium dysfunction. Herein, we propose a pulmonary endothelium-targeted codelivery system of anti-inflammatory indomethacin (IND) and antioxidant superoxide dismutase (SOD) by assembling the biopharmaceutical SOD onto the "vector" of rod-like pure IND crystals, followed by coating with anti-ICAM-1 antibody (Ab) for targeting endothelial cells. The codelivery system has a 237 nm diameter in length and extremely high drug loading of 39% IND and 2.3% SOD. Pharmacokinetics and biodistribution studies demonstrate the extended blood circulation and the strong pulmonary accumulation of the system after intravenous injection in the lipopolysaccharide (LPS)-induced inflammatory murine model. Particularly, the system allows a robust capacity to target pulmonary endothelium mostly due to the rod-shape and Ab coating effect. In vitro , the preparation shows the synergistic anti-inflammatory and antioxidant effects in LPS-activated endothelial cells. In vivo , the preparation exhibits superior pharmacodynamic efficacy revealed by significantly downregulating the inflammatory/oxidative stress markers, such as TNF- α , IL-6, COX-2, and reactive oxygen species (ROS), in the lungs. In conclusion, the codelivery system based on rod-like pure crystals could well target the pulmonary endothelium and effectively alleviate lung inflammation. The study offers a promising approach to combat pulmonary endothelium-associated diseases. The codelivery system Ab-INRplex attaches to the ICAM-1 overexpressed on inflamed endothelial cells, and synergistically relieves the inflammation by inhibiting COX-2 and reactive oxygen species production. [Display omitted] [ABSTRACT FROM AUTHOR]

A key parameter for the success of most cellular implants is the formation of a complete and comprehensive intra-implant vessel network. Pre-vascularization, the generation of vessel structures in vitro prior to transplantation, provides accelerated implant perfusion via anastomosis, but scalability and ease of integration hinder clinical translation. For fibrin-based vasculogenesis approaches, the remodeling and degradation of the fragile, hydrogel matrix during the formation of vessel-like structures results in rapid, cell-mediated construct compaction leading to dense, capillary-like structures with ineffective network coverage. To resolve these challenges, vasculogenic hydrogels were embedded within a highly porous, biostable three-dimensional (3D) polydimethylsiloxane (PDMS) scaffold. Using reverse-casting of 3D-printed molds, scaffolds exhibited highly interconnected and reproducible pore structures. Pore size was optimized via in vivo screening of intra-device angiogenesis. The inclusion of the PDMS frame with vasculogenic hydrogels significantly reduced fibrin compaction in vitro , resulting in easily manipulated constructs with predictable dimensionality and increased surface area compared to fibrin hydrogel alone. Globally, vascular morphogenesis was altered by the PDMS frame, with significantly larger and less dense network structures. Vasculogenic proteomic evaluation showed a temporal impact of the addition of the PDMS frame, indicating altered cellular proliferation and migration signaling. This work establishes a platform for improving the generation of translational pre-vascularized networks for greater flexibility to meet the needs of clinically scaled, engineered tissues. Competent intra-implant vascularization is a significant issue hindering the success of engineered tissues. Pre-vascularization approaches, whereby a vascular network is formed in vitro and subsequently implanted into the host to anastomose, is a promising approach but it is limited by the compacted, dense, and poorly functional microcapillary structures typically formed using soft hydrogels. Herein, we have uniquely addressed this challenge by adding a 3D printed PDMS-based open framework structure that serves to prevent hydrogel compaction. Globally, we observed distinct differences in overall construct geometry, vascular network density, compaction, and morphogenesis, indicating that this PDMS framework lead to elevated maturity of this in vitro network while retaining its global dimensions. Overall, this novel approach elevates the translational potential of pre-vascularized constructs. [Display omitted] [ABSTRACT FROM AUTHOR]

The Descemet Membrane Endothelial Keratoplasty (DMEK) procedure for corneal transplantation is challenging due to the need to unscroll the donor graft within the recipient's eye. This process of unscrolling is complex, time-consuming, leads to a loss of endothelial cells and, most importantly, can negatively impact the graft's adhesion and integration with the host tissue after surgery. This problem is particularly evident when the graft is young. However, the physics behind this scrolling is not well understood, and therefore no sustainable solution is attained. Here, we propose that the concentration gradient of the medium used during transplant leads to a displacement gradient across the graft thickness, resulting in an out-of-plane folding or scrolling of the graft tissue. Using chitosan bilayer-based experimental models, it is experimentally demonstrated that this diffusion-coupled-deformation phenomenon can successfully explain why younger donor grafts tend to scroll tighter than older ones. Most importantly, we illustrate here through experiments that the medium can be engineered to reduce the scroll tightness and thus reduce the surgical inconveniences and improve post-transplant recovery. This paper addresses a major issue that surgeons face while doing Descemet Membrane Endothelial Keratoplasty (DMEK) in unscrolling grafts during the graft insertion procedure. The currently used tapping method to unscroll the graft inside the patient's eye significantly reduces endothelial cell count, thus affecting its lifetime. Surprisingly, the physics behind graft scrolling is not well understood, so no sustainable solutions are proposed by the medical community. In this work, we present the underlying mechanism of DMEK graft scroll and illustrate experimentally the reason for scroll tightness through a chitosan bilayer based experiment model. Most importantly, we have successfully demonstrated that the preserving medium of the grafts can be engineered to reduce scroll tightness. [Display omitted] [ABSTRACT FROM AUTHOR]

Tissue regeneration/fibrosis after injury is intricately regulated by the immune cascade reaction and extracellular matrix (ECM). Dysregulated cascade signal could jeopardize tissue homeostasis leading to fibrosis. Bioactive scaffolds mimicking natural ECM microstructure and chemistry could regulate the cascade reaction to achieve tissue regeneration. The current study constructed an ECM-engineered micro/nanofibrous scaffold using self-assembled nanofibrous collagen and decorin (DCN)-loaded microfibers to regulate the immune cascade reaction. The ECM-engineered scaffold promoted anti-inflammatory and pro-regenerative effects, M2 polarization of macrophages, by nanofibrous collagen. The ECM-engineered scaffold could release DCN to inhibit inflammation-associated fibrous angiogenesis. Yet, to prevent excessive M2 activity leading to tissue fibrosis, controlled release of DCN was expected to elicit M1 activity and achieve M1/M2 balance in the repair process. Regulated cascade reaction guided favorable crosstalk between macrophages, endothelial cells and fibroblasts by proximity. Additionally, decorin could also antagonize TGF-β1 via TGF-β/Smad3 pathway to suppress fibrotic activity of fibroblasts. Hence, ECM-engineered scaffolds could exert effective regulation of the immune cascade reaction by microstructure and DCN release and achieve the balance between tissue fibrosis and regeneration. With the incidence of up to 74.6%, failed back surgery syndrome (FBSS) has been a lingering issue in spine surgery, which poses a heavy socio-economic burden to society. Epidural fibrosis is believed to be responsible for the onset of FBSS. Current biomaterial-based strategies treating epidural fibrosis mainly rely on physical barriers and unidirectional suppression of inflammation. Regulation of the immune cascade reaction for inhibiting fibrosis has not been widely studied. Based on the simultaneous regulation of M1/M2 polarization and intercellular crosstalk, the ECM-engineered micro/nanofibrous scaffolds constructed in the current study could exert an immune cascade effect to coordinate tissue regeneration and inhibit fibrosis. This finding makes a significant contribution in the development of a treatment for epidural fibrosis and FBSS. [Display omitted] [ABSTRACT FROM AUTHOR]

Cardiovascular diseases, especially atherosclerosis, are the leading cause of human mortality in Indonesia. Ipomoea batatas (L.) is a food plant used in Indonesian traditional medicine to treat cardiovascular diseases and related conditions. We assessed the anti-atherosclerotic activity of the aqueous extract of I. batatas leaves in a rat model of high-fat diet-induced atherosclerosis and its mechanism. The presence of amino acid content in the I. batatas L. purple variant was determined by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Thirty male Wistar rats were divided into five groups (n=6/group), i.e., standard diet group (SD), high-fat diet group (HF), and HF plus I. batatas L. extracts orally (625; 1,250; or 2,500 mg/kg) groups. The numbers of macrophages and aortic wall thickness were analyzed histologically. Immunohistochemical analyses were performed to assess foam cells-oxidized low-density lipoprotein (oxLDL), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF) expression in the aorta. LC-HRMS analysis showed nine amino acid content were identified from I. batatas L. In vivo study revealed that oral administration of I. batatas L. leaf extract alleviated foam cells-oxLDL formation and aortic wall thickness caused by high-fat diet atherosclerosis rats. Further, I. batatas L. leaf extract promoted the number of macrophages and modulated VEGF and eNOS expression in the aorta. I. batatas L. leaf extract shows a positive anti-atherosclerosis effect. Furthermore, the mechanism may promote the macrophages, eNOS, VEGF expressions, and inhibition of foam cells-oxLDL formation and aortic wall thickness with the best dosage at 2,500 mg/kg. This could represent a novel approach to prevent cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Introduction: Cerebrovascular pathology is an early and causal hallmark of Alzheimer's disease (AD), in need of effective therapies. Methods: Based on the success of our previous in vitro studies, we tested for the first time in a model of AD and cerebral amyloid angiopathy (CAA), the carbonic anhydrase inhibitors (CAIs) methazolamide and acetazolamide, Food and Drug Administration–approved against glaucoma and high‐altitude sickness. Results: Both CAIs reduced cerebral, vascular, and glial amyloid beta (Aβ) accumulation and caspase activation, diminished gliosis, and ameliorated cognition in TgSwDI mice. The CAIs also improved microvascular fitness and induced protective glial pro‐clearance pathways, resulting in the reduction of Aβ deposition. Notably, we unveiled that the mitochondrial carbonic anhydrase‐VB (CA‐VB) is upregulated in TgSwDI brains, CAA and AD+CAA human subjects, and in endothelial cells upon Aβ treatment. Strikingly, CA‐VB silencing specifically reduces Aβ‐mediated endothelial apoptosis. Discussion: This work substantiates the potential application of CAIs in clinical trials for AD and CAA. [ABSTRACT FROM AUTHOR]

BACKGROUND: Rare genetic variants and genetic variation at loci in an enhancer in SOX17 (SRY-box transcription factor 17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutations in SOX17 in PAH pathogenesis has not been reported. METHODS: SOX17 expression was evaluated in the lungs and pulmonary endothelial cells (ECs) of patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion were generated to determine the role of SOX17 deficiency in the pathogenesis of PAH. Human pulmonary ECs were cultured to understand the role of SOX17 deficiency. Single-cell RNA sequencing, RNA-sequencing analysis, and luciferase assay were performed to understand the underlying molecular mechanisms of SOX17 deficiency-induced PAH. E2F1 (E2F transcription factor 1) inhibitor HLM00 6474 was used in EC-specific Sox17 mice. RESULTS: SOX17 expression was downregulated in the lung and pulmonary ECs from patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion induced spontaneously mild pulmonary hypertension. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced pulmonary hypertension in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and antiapoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP (bone morphogenetic protein) signaling. E2F1 signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction. Pharmacological inhibition of E2F1 in Sox17 EC-deficient mice attenuated pulmonary hypertension development. CONCLUSIONS: Our study demonstrated that endothelial SOX17 deficiency induces pulmonary hypertension through E2F1. Thus, targeting E2F1 signaling represents a promising approach in patients with PAH. [ABSTRACT FROM AUTHOR]

BACKGROUND: Left ventricular hypertrophy is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time develops maladaptive pathology partly due to microvascular rarefaction and impaired coronary angiogenesis. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate coronary angiogenesis, leading to heart failure, are not well known. METHODS: We subjected endothelial-specific knockout mice of mechanically activated ion channel, TRPV4 (transient receptor potential cation channel subfamily V member 4; TRPV4ECKO) to pressure overload via transverse aortic constriction and examined cardiac function, cardiomyocyte hypertrophy, cardiac fibrosis, and apoptosis. Further, we measured microvascular density and underlying TRPV4 mechanotransduction mechanisms using human microvascular endothelial cells, extracellular matrix gels of varying stiffness, unbiased RNA sequencing, small interfering RNA, Western blot, quantitative-PCR, and confocal immunofluorescence techniques. RESULTS: We demonstrate that endothelial-specific deletion of TRPV4 preserved cardiac function, cardiomyocyte structure, and reduced cardiac fibrosis compared with TRPV4lox/lox mice, 28 days post-transverse aortic constriction. Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of proangiogenic factors (VEGFα [vascular endothelial growth factor a], NOS3 [nitric oxide synthase 3], and FGF2 [fibroblast growth factor 2]) with concomitant increase in microvascular density in TRPV4ECKO hearts after transverse aortic constriction compared with TRPV4lox/lox. Further, an increased expression of VEGFR2 (vascular endothelial growth factor receptor 2) and activation of the YAP (yes-associated protein) pathway were observed in TRPV4ECKO hearts. Mechanistically, we found that downregulation of TRPV4 in endothelial cells induced matrix stiffness-dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/large tumor suppressor kinase pathway. CONCLUSIONS: Our results suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling endothelial TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis. [ABSTRACT FROM AUTHOR]

The utilization of Poly-L-lactic acid (PLLA) as a biodegradable material for biovascular scaffolds (BVS) stems from its superior mechanical attributes, biodegradability, and biocompatibility. However, PLLA's challenges, such as its lack of active surface functional groups and high hydrophobicity, impede endothelial cells' (ECs) attachment and growth. These factors subsequently amplify the risk of thrombosis and restenosis in PLLA implants. To counteract this problem, we've adapted the polydopamine (PDA) coating technique, a technique commonly used for metal stents. This method has demonstrated potential for promoting EC adhesion and proliferation on PLLA surfaces. Our study involved the application of PDA coating to PLLA, which resulted in improved EC attachment and proliferation while simultaneously inhibiting macrophage activation and the release of the inflammatory factor TNF-α. Additionally, the reactive functional groups on the PDA-coated surface provide a reactive platform for immobilizing therapeutic molecules onto PLLA, thereby further enhancing the biocompatibility of PLLA cardiovascular stents. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated the critical role of cardiac-resident macrophages (cMacs) in the maintenance of physiological homeostasis. However, recruitment of circulating monocyte-derived macrophages decreases cMac levels post-myocardial infarction (MI). Transplanting cMacs is not an ideal option due to their low survival rates and the risk of immunological rejection. However, extracellular vesicle therapy has the potential to provide a feasible and safe alternative for cardiac repair. In this study, cell membrane-modified extracellular vesicles (MmEVs) were developed for heart repair by modifying cMac-derived extracellular vesicles (mEVs) with monocyte membranes, resulting in immune evasion and sequential targeted localization to damaged regions through expression of CD47 on MmEVs and strong affinity between monocyte membrane proteins and CCL2. Additionally, to fully exploit the potential clinical application of MmEVs and achieve a better curative effect, thymosin β4 (Tβ4) was loaded into the nanoparticles, resulting in Tβ4-MmEVs. In vitro experiments indicated that both the MmEVs and Tβ4-MmEVs promoted cardiomyocyte proliferation and endothelial cell migration. Animal experiments suggested that MI mice treated with MmEVs and Tβ4-MmEVs exhibited reduced myocardial fibrosis and increased vascular density compared to the control group. Thus, we posit that these targeted nanoparticles hold significant potential for MI adjuvant therapy and may open new avenues for cardiac repair and regeneration. Extracellular vesicles (EVs) derived from bioactive parent cell sources involved in pathological and repair processes for cardiovascular disease have emerged as a compelling strategy for regenerative therapy. In this study, we constructed monocyte membrane-modified extracellular vesicles loaded with a drug (Tβ4-MmEVs) for heart repair that exhibit extraordinary abilities of immune evasion and sequential localization to damaged regions owing to the presence of CD47 and the strong affinity between monocytes and damaged cardiomyocytes and endothelial cells. The bioactivities of Tβ4-MmEVs on enhancing cardiomyocyte and endothelial cell proliferation were validated both in vitro and in vivo. Effective development and implementation of therapeutically membrane-modified nanoparticles from homologous origins can provide a reference for adjuvant therapy in clinical MI management. [Display omitted] [ABSTRACT FROM AUTHOR]

While hyaluronic acid (HA)-based hydrogels have been used clinically for decades, the mechanisms by which HA exerts molecular weight-dependent bioactivity and how chemical modification and crosslinking may affect molecular weight-dependent bioactivity remain poorly understood. This knowledge gap presents a significant barrier to designing HA hydrogels with predictable bioactivities. As HA has been widely reported to have molecular weight-dependent effects on endothelial cells (ECs), we investigated how the molecular weight of HA in either soluble or crosslinked forms affects angiogenesis and interrogated CD44 clustering on the surface of endothelial cells as a candidate mechanism for these affects. Using soluble HA, our results show high molecular weight (HMW) HA, but not low molecular weight (LMW) HA, increased viability and tube formation in cultured human cerebral microvascular ECs (HCMVECs). No size of HA affected proliferation. When HCMVECs were cultured with crosslinked HA of varying molecular weights in the form of HA-based microporous annealed particle scaffold (HMAPS), the cell response was comparable to when cultured with soluble HA. Similarly, when implanted subcutaneously, HMAPS with HMW HA were more vascularized than those with LMW HA. We also show that antibody-mediated CD44 clustering resulted in HCMVECs with increased viability and tube-like structure formation in a manner comparable to exposure to HMW HA, suggesting that HMW acts through CD44 clustering. Biomaterials based on hyaluronic acid (HA), a bioactive extracellular matrix polysaccharide, have been used in clinical products for several years. Despite the knowledge that HA molecular weight heavily influences its bioactivity, molecular weight has been largely ignored in the development of HA-based biomaterials. Given the high viscosity of high molecular weight HA typically found in native tissues, lower molecular weight polysaccharides have been used most commonly for biomaterial fabrication. By comparing the ability of injectable, microporous annealed particle scaffolds (MAPS) fabricated from variably sized HA to promote angiogenesis, this study demonstrates that MAPS with high molecular weight HA better support vascularization, likely through an unique ability to induce clustering of CD44 receptors on endothelial cells. [Display omitted] [ABSTRACT FROM AUTHOR]

BACKGROUND: Type 1 diabetes (T1D) is a major cause of endothelial dysfunction. Although cellular bioenergetics has been identified as a new regulator of vascular function, whether glycolysis, the primary bioenergetic pathway in endothelial cells (EC), regulates vascular tone and contributes to impaired endothelium-dependent relaxation (EDR) in T1D remains unknown. METHODS: Experiments were conducted in Akita mice with intact or selective deficiency in EC PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), the main regulator of glycolysis. Seahorse analyzer and myography were employed to measure glycolysis and mitochondrial respiration, and EDR, respectively, in aortic explants. EC PFKFB3 (Ad-PFKFB3) and glycolysis (Ad-GlycoHi) were increased in situ via adenoviral transduction. RESULTS: T1D increased EC glycolysis and elevated EC expression of PFKFB3 and NADPH oxidase Nox1 (NADPH oxidase homolog 1). Functionally, pharmacological and genetic inhibition of PFKFB3 restored EDR in T1D, while in situ aorta EC transduction with Ad-PFKFB3 or Ad-GlycoHi reproduced the impaired EDR associated with T1D. Nox1 inhibition restored EDR in aortic rings from Akita mice, as well as in Ad-PFKFB3-transduced aorta EC and lactate-treated wild-type aortas. T1D increased the expression of the advanced glycation end product precursor methylglyoxal in the aortas. Exposure of the aortas to methylglyoxal impaired EDR, which was prevented by PFKFB3 inhibition. T1D and exposure to methylglyoxal increased EC expression of HIF1α (hypoxia-inducible factor 1α), whose inhibition blunted methylglyoxal-mediated EC PFKFB3 upregulation. CONCLUSIONS: EC bioenergetics, namely glycolysis, is a new regulator of vasomotion and excess glycolysis, a novel mechanism of endothelial dysfunction in T1D. We introduce excess methylglyoxal, HIF1α, and PFKFB3 as major effectors in T1D-mediated increased EC glycolysis. [ABSTRACT FROM AUTHOR]

A complete in vitro skin model, containing resident cell types is needed to understand physiology and to consider the role of immune and endothelial cells in dermal drug testing. In this study, a cell extraction technique was developed to isolate resident skin cells from the same human donor while preserving the immune and endothelial cells. Then those cells were used to reconstruct an autologous, vascularized, and immunocompetent Tissue-Engineered Skin model, aviTES. Phenotypic characterization of the viable cells was performed on freshly isolated cells and after thawing through flow cytometry. Dermal cell extracts were characterized as fibroblasts, endothelial and immune cells, and the average amount of each cell type represents 4, 0.5, and 1 million viable cells per g of the dermis, respectively. The 3D models, TES and aviTES, were characterized by a fully differentiated epidermis that showed an increase in the presence of Ki67+ cells in the basolateral layer of the aviTES model. Capillary-like network formation, through the self-assembly of endothelial cells, and the presence of functional immune cells were identified through immunofluorescence staining in aviTES. In addition, the aviTES model was immunocompetent, as evidenced by its capacity to increase the production of pro-inflammatory cytokines TNF-α, MIP-1α, and GM-CSF following LPS stimulation. This study describes an autologous skin model containing a functional resident skin immune system and a capillary network. It provides a relevant tool to study the contribution of the immune system to skin diseases and inflammatory responses and to investigate resident skin cell interactions and drug development. There is an urgent need for a complete in vitro skin model containing the resident cell types to better understand the role of immune and endothelial cells in skin and to be able to use it for drug testing. Actual 3D models of human skin most often contain only fibroblasts and keratinocytes with a limited number of models containing endothelial cells or a limited variety of immune cells. This study describes an autologous skin model containing a functional resident skin immune system and a capillary network. It provides a relevant tool to study the contribution of the immune system to skin diseases and inflammatory responses and to investigate interactions between resident skin cell, improving our capacity to develop new drugs. [Display omitted] [ABSTRACT FROM AUTHOR]

Endotheliopathy plays a role in the development of acute kidney and lung injury in COVID-19, probably due to inflammation, endothelial permeability, vascular leakage and edema formation. This study examined alterations in the circulation of patients with mild and severe COVID-19 on in vitro endothelial permeability and its relation to the endothelial angiopoietin/Tie2 system, which is involved in the regulation of endothelial permeability. Plasma was obtained from COVID-19 patients admitted to the ward (n = 14) or ICU (n = 20) at admission and after 1 and 2 weeks and healthy controls (n = 5). Human kidney and lung endothelial cells were exposed to patient plasma and treated with recombinant angiopoietin-1. In vitro endothelial barrier function was assessed using electric cell-substrate impedance sensing. Circulating markers of the angiopoietin/Tie2 system, endothelial dysfunction and glycocalyx degradation were measured by ELISA. Plasma from COVID-19 patients reduced endothelial resistance compared to healthy controls, but COVID-19 plasma-induced drop in endothelial resistance did not differ between ward and ICU patients. Circulating angiopoietin-2, soluble Tie2 and soluble Tie1 levels increased over time in ICU patients, whereas levels remained stable in ward patients. The increase in angiopoietin-2 was able to predict 90-day mortality (AUC = 0.914, p < 0.001). Treatment with recombinant angiopoietin-1 did not restore COVID-19 plasma-induced hyperpermeability. In conclusion, these results suggest that indirect effects of the virus represented in the circulation of COVID-19 patients induced endothelial hyperpermeability irrespective of disease severity and changes in the endothelial angiopoietin/Tie2 system. Nonetheless, angiopoietin-2 might be of interest in the context of organ injury and patient outcome in COVID-19. [ABSTRACT FROM AUTHOR]

A 12-y-old, male Dachshund was presented for elective orchiectomy. The testes were of normal size. The left testis had numerous dark-red, blood clot–like foci within the vaginal tunic over the pampiniform plexus, epididymis, and testis. Histologically, the red foci were limited to the vaginal tunic and consisted of disorderly growing, variably sized, thin-walled blood vessels lined by a single layer of endothelial cells without mitoses and supported by a thin layer of pericytes. The blood vessels were distended by erythrocytes without thrombus formation. Endothelial cells had cytoplasmic immunolabeling for CD31; pericytes had strong cytoplasmic immunolabeling for α–smooth muscle actin. Our case of subclinical unilateral vascular hamartomas of the vaginal tunic in a dog has not been reported previously in domestic animals or humans, to our knowledge. [ABSTRACT FROM AUTHOR]

MicroRNAs play a crucial role in regulating cartilage extracellular matrix (ECM) metabolism and are being explored as potential therapeutic targets for osteoarthritis (OA). The present study indicated that microRNA-224-5p (miR-224-5p) could balance the homeostasis of OA via regulating cartilage degradation and synovium inflammatory simultaneously. Multifunctional polyamidoamine dendrimer with amino acids used as efficient vector to deliver miR-224-5p. The vector could condense miR-224-5p into transfected nanoparticles, which showed higher cellular uptake and transfection efficiency compared to lipofectamine 3000, and also protected miR-224-5p from RNase degradation. After treatment with the nanoparticles, the chondrocytes showed an increase in autophagy rate and ECM anabolic components, as evidenced by the upregulation of autophagy-related proteins and OA-related anabolic mediators. This led to a corresponding inhibition of cell apoptosis and ECM catabolic proteases, ultimately resulting in the alleviation of ECM degradation. In addition, miR-224-5p also inhibited human umbilical vein endothelial cells angiogenesis and fibroblast-like synoviocytes inflammatory hyperplasia. Integrating the above synergistic effects of miR-224-5p in regulating homeostasis, intra-articular injection of nanoparticles performed outstanding therapeutic effect by reducing articular space width narrowing, osteophyte formation, subchondral bone sclerosis and inhibiting synovial hypertrophy and proliferation in the established mouse OA model. The present study provides a new therapy target and an efficient intra-articular delivery method for improving OA therapy. Osteoarthritis (OA) is the most prevalent joint disease worldwide. Gene therapy, which involves delivering microRNAs, has the potential to treat OA. In this study, we demonstrated that miR-224-5p can simultaneously regulate cartilage degradation and synovium inflammation, thereby restoring homeostasis in OA gene therapy. Moreover, compared to traditional transfection reagents such as lipofectamine 3000, G5-AHP showed better efficacy in both microRNA transfection and protection against degradation due to its specific surface structure. In summary, G5-AHP/miR-224-5p was developed to meet the clinical needs of OA patients and the high requirement of gene transfection efficiency, providing a promising paradigm for the future application and development of gene therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) can respond to various mechanical cues such as shear stress and substrate stiffness. In the human brain, the compromised barrier function of the BBB is closely associated with a series of neurological disorders that are often also accompanied by the alteration of brain stiffness. In many types of peripheral vasculature, higher matrix stiffness decreases barrier function of endothelial cells through mechanotransduction pathways that alter cell-cell junction integrity. However, human brain endothelial cells are specialized endothelial cells that largely resist changes in cell morphology and key BBB markers. Therefore, it has remained an open question how matrix stiffness affects barrier integrity in the human BBB. To gain insight into the effects of matrix stiffness on BBB permeability, we differentiated brain microvascular endothelial-like cells from human induced pluripotent stem cells (iBMEC-like cells) and cultured the cells on extracellular matrix-coated hydrogels of varying stiffness. We first detected and quantified the junction presentation of key tight junction (TJ) proteins. Our results show matrix-dependent junction phenotypes in iBMEC-like cells, where cells on softer gels (1 kPa) have significantly lower continuous and total TJ coverages. We also determined that these softer gels also lead to decreased barrier function in a local permeability assay. Furthermore, we found that matrix stiffness regulates the local permeability of iBMEC-like cells through the balance of continuous ZO-1 TJs and no junction regions ZO-1 in tricellular regions. Together, these findings provide valuable insights into the effects of matrix stiffness on TJ phenotypes and local permeability of iBMEC-like cells. Brain mechanical properties, including stiffness, are particularly sensitive indicators for pathophysiological changes in neural tissue. The compromised function of the blood-brain barrier is closely associated with a series of neurological disorders often accompanied by altered brain stiffness. In this study, we use polymeric biomaterials and provide new evidence that biomaterial stiffness regulates the local permeability in iPSC-derived brain endothelial cells in tricellular regions through the tight junction protein ZO-1. Our findings provide valuable insights into the changes in junction architecture and barrier permeability in response to different substrate stiffnesses. Since BBB dysfunction has been linked to many diseases, understanding the influence of substrate stiffness on junction presentations and barrier permeability could lead to the development of new treatments for diseases associated with BBB dysfunction or drug delivery across BBB systems. [Display omitted] [ABSTRACT FROM AUTHOR]